Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling

Journal of Immunological Methods 271 (2002) 185 – 201 www.elsevier.com/locate/jim

Protocol

Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling Caroline Gilbert a,b, Emmanuelle Rollet-Labelle a,b, Adriana C. Caon c, Paul H. Naccache a,b,* a

Centre de Recherche en Rhumatologie et Immunologie, CIHR group on the Molecular Mechanisms of Inflammation, Centre de Recherche du CHUL, Laval University, Ste.-Foy, Que´bec, Canada b Department of Medicine, Faculty of Medicine, Laval University, Ste.-Foy, Que´bec, Canada c Department of Molecular Biosciences, University of Adelaide, North Terrace, Adelaide, Australia Received 30 August 2002; accepted 5 September 2002

Abstract In stimulated neutrophils, the majority of tyrosine-phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells that are lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserve and solubilize tyrosinephosphorylated proteins but also retain their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilization of a significant fraction of tyrosinephosphorylated proteins. Furthermore, we observed in neutrophils in which CD32 was cross-linked that the tyrosine kinase activity of Lyn was enhanced in the soluble fraction recovered from the hypertonic lysis but not in that derived from the first hypotonic lysis. Furthermore, we detected tyrosine kinase activity and the presence of the tyrosine kinase Syk in association with CD32 in the soluble hypertonic lysis fraction. This fraction also contained most of the tyrosine-phosphorylated proteins including Cbl, Syk and CD32 itself. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cbl; CD32; Detergent insolubility; Fcg receptors; Kinase activity; Lyn; Monosodium urate monohydrate crystals; Neutrophil; Phagocytosis; Signal transduction; Syk; Tyrosine phosphorylation

Abbreviations: HLB, hypotonic lysis buffer; HyperLB, hypertonic lysis buffer; ILB, isotonic lysis buffer; LB, lysis buffer; RIPA, rapid immunoprecipitation assay; SB, sample buffer. * Corresponding author. CHUL, Room T1-49, 2705 Boulevard Laurier, Ste.-Foy, Que´bec, Canada G1V 4G2. Tel.: +1-418-6542772; fax: +1-418-654-2765. E-mail address: [email protected] (P.H. Naccache).

1. Background Neutrophils play a crucial role in host defense against injury and infection as well as in the inflammatory response (Smith, 1994) by virtue of their ability to mount a series of effector responses. Neutrophils respond to a wide variety of agonists and one

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 3 4 7 - 2

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of the earliest events observed upon stimulation of neutrophils is an increase in the level of tyrosine phosphorylation of a number of proteins (Rollet et al., 1994). The mechanisms linking neutrophil surface receptors to tyrosine kinase signaling are, however, incompletely understood. Immunoprecipitation coupled with immunoblotting is one of the most commonly used immunochemical techniques for studying tyrosine phosphorylation-dependent pathways. This technique can potentially determine several important characteristics of molecules under examination such as their presence and quantities, their relative molecular weights, their rates of synthesis or degradation, and the presence of certain post-translational modifications. It can also provide information about interactions between proteins, nucleic acids and other ligands. These protocols depend on lysing the cells under mild conditions (usually RIPA buffers) containing cocktails of protease and phosphatase inhibitors. These lysates are then centrifuged and the supernatants are used for subsequent immunoprecipitations. It is commonly observed, however, that the profile of tyrosine-phosphorylated proteins of whole neutrophils is rapidly lost or artificially increased upon lysing the cells in classical RIPA buffers (Al-Shami et al., 1997b; Naccache et al., 1997). An alternative assay involving lysis under denaturing conditions was previously developed to preserve the phosphorylation levels in neutrophil lysates (Al-Shami et al., 1997b). Although this protocol allows the identification of tyrosine-phosphorylated proteins (Al-Shami et al., 1997a; Barabe´ et al., 1998; Khamzina and Borgeat, 1998; RolletLabelle et al., 2000; Gilbert et al., 2001), it disrupts protein – protein interactions and inactivates most enzymatic activities. Most studies analyze the soluble fractions following lysis in RIPA buffers while discarding the insoluble fractions. However, the formation of detergent-resistant membrane structures (DRM, DIGs or lipids rafts) into which tyrosine-phosphorylated proteins and tyrosine kinases tend to concentrate after receptor stimulation has been welldescribed in a range of cell types (Brown and London, 1998; Simons, 2000) including neutrophils (Zhou et al., 1995; Yan et al., 1996; Barabe´ et al., 2002). The function of these detergent-insoluble

structures appears to be related to signal transmission and/or membrane trafficking. Accordingly, tyrosine kinase activities were found to be increased and concentrated in these insoluble fractions upon ligation of phagocytic receptors in adherent human neutrophils (Zhou et al., 1995; Yan et al., 1996). Accurate analysis of tyrosine phosphorylation events in these structures depends on the preparation of stable cell lysates that can be used as starting material for their analysis by immunoprecipitation. In the present study, a method designed to reach the above aims, based on sequential cell lysis under conditions of increasing tonicity, is described. Firstly, we show the distribution and preservation of tyrosinephosphorylated substrates in human neutrophil lysates prepared under native conditions. The results obtained illustrate the inadequacy of using standard cell lysates prepared under native conditions as starting material in subsequent immunoprecipitation or co-immunoprecipitation protocols. Secondly, we observe, using our modified protocol, that the tyrosine kinase activity of Lyn is specifically increased in the hypertonic lysis fractions. This fraction is enriched in GM1 ganglioside and cytoskeletal components. Thirdly, we show in the hypertonic lysis fractions an association between the tyrosine kinase Syk and cross-linked CD32. Finally, we also observe that Cbl, Syk and CD32 were highly tyrosine-phosphorylated, specifically in the soluble fraction of the hypertonic lysis step. The use of this alternative protocol emphasizes the critical importance of the complete monitoring of the tyrosine phosphorylation status as well as of the solubility and, when applicable, the enzymatic activity of each target molecule, in response to each agonist.

2. Type of research (i) Signal transduction. (ii) Analysis of tyrosine phosphorylation profiles and identification of tyrosine-phosphorylated substrates. (iii) Distribution of tyrosine-phosphorylated proteins between soluble and insoluble fractions. (iv) Measurements of tyrosine kinase activities upon stimulation in the soluble and insoluble fractions.

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3. Time required (i) (ii) (iii) (iv) (v) (vi)

Cell isolation (2 h) Cell stimulation and lysis (15 – 45 min) Lysate immunoprecipitation (3 h) Kinase activity (10 – 60 min) Electrophoresis (5 h or O/N) Transfer to Immobilon PVDF membranes (3 h or O/N) (vii) Immunoblot (3– 4 h).

4. Materials 4.1. Preparation of cells HBSS 1  solution (Wisent Canadian Laboratories, St.-Bruno, Que´bec, Canada) containing 1.6 mM CaCl2 + 10 mM HEPES 2% of dextran solution (Pharmacia Biotech, Dorval, Que´bec, Canada in HBSS 1  ) Ficoll Paque (Wisent Canadian Laboratories) 10% trypan blue solution (Sigma-Aldrich Canada, Oakville, ON, Canada). 4.2. Preparation of buffers 2  Laemmli’s sample buffer (2  SB) 125 mM Tris – HCl, pH 6.8 8% SDS 10% h-mercaptoethanol 17.5% glycerol 5 mM Na3VO4 (stock solution is prepared as previously described (Papavassiliou, 1994)) 20 mM p-nitrophenylphosphate 20 Ag/ml leupeptin 20 Ag/ml aprotinin 0.0025% bromophenol blue 2  lysis buffer (2  LB) 125 mM Tris – HCl, pH 6.8 4% SDS 3% h-mercaptoethanol 17.5% glycerol 5 mM Na3VO4 20 Ag/ml leupeptin 20 Ag/ml aprotinin 0.0025% bromophenol blue

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Hypotonic lysis buffer (HLB) 0.1% NP-40 (10% NP-40, Calbiochem, La Jolla, CA, USA) 20 mM Tris – HCl, pH 7.5 at 4 jC 10 mM NaCl 1 mM EDTA 2 mM Na3VO4 10 Ag/ml aprotinin 10 Ag/ml leupeptin 2 mM PMSF (optional, to be added immediately prior to lysing the cells) 50 Ag/ml soybean trypsin inhibitor 3 mM DFP (to be added just prior to lysing the cells. Take care to discard the tips in a tube containing 1N NaOH in order to neutralize the DFP) Isotonic lysis buffer (ILB) 1% NP-40 (100% NP-40, Calbiochem) 20 mM Tris – HCl, pH 7.5 at 4 jC 137 mM NaCl 1 mM EDTA 2 mM Na3VO4 10 Ag/ml aprotinin 10 Ag/ml leupeptin 2 mM PMSF 50 Ag/ml soybean trypsin inhibitor Hypertonic lysis buffer (HyperLB) 1% NP-40 (from 100% NP-40, Calbiochem) 20 mM Tris – HCl, pH 7.5 at 4 jC 400 mM NaCl 1 mM EDTA 2 mM Na3VO4 10 Ag/ml aprotinin 10 Ag/ml leupeptin 2 mM PMSF 50 Ag/ml soybean trypsin inhibitor Immunoprecipitation wash buffer (IPPWashB) 1% NP-40 (from 100% NP-40, Calbiochem) 20 mM Tris – HCl, pH 7.5 at 4 jC 137 mM NaCl 1 mM EDTA (omit in the last wash if measuring kinase activity) Kinase buffer (1  KB) 50 mM HEPES, pH 7.5 10 mM MgCl2 3 mM MnCl2 50 AM ATP (prepared from powder just prior to starting the kinase assay)

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0.5 Ag of SAM68 – GST per immunoprecipitation. 4.3. Preparation of polyacrylamide gel (7.5 – 20% SDS-PAGE) 30% acrylamide solution 0.3% bisacrylamide solution This provides optimal separation for a wide range of molecular weight proteins (8 –150 kDa). 4.4. Preparation of transfer and blotting solutions Transfer buffer 25 mM of Tris base 192 mM glycine 20% (v/v) methanol Ponceau 1% 1.0 g Ponceau S 1.0 ml glacial acetic acid 99.0 ml H2O Tris buffer/Tween 20 (TBS) 25 mM Tris HCl, pH 8.0 190 mM NaCl 0.15% (v/v) Tween 20 Blocking solution 2% (v/v) gelatin 100 ml TBS Microwave for 30 s to dissolve gelatin or warm in a water bath or on a hot plate. Cool to 37 jC before use. 4.5. Antibodies (1) The polyclonal antibodies, anti-Cbl (sc-170, 1:1000), anti-Lyn (sc-15, 1:2000) and antiSAM68 (sc-333, 1:1000) are purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-mouse heavy/light chains (315-005-048) is purchased from Jackson Immune Research (Mississauga, ON, Canada). (2) The monoclonal antibodies anti-Syk (MAB88906, 1:1000), anti-phosphotyrosine (UBI 05-321, clone 4G10, 1:4000) are purchased from Chemicon International (Missisauga, ON, Canada) and Upstate Biotechnology (Lake Placid, NY, USA), respectively. The monoclonal antibodies antiEzrin (E53020, 1:5000), anti-p62 nucleoporin

(N43620, 1:1000) and the organelle Sampler kit antibodies (611436) are obtained from Transduction Laboratories (Lexington, KY, USA). (3) An anti-FcgRII polyclonal antiserum against the cytoplasmic tail of CD32 (CT-10) is generated in our laboratory as described previously (Ibarrola et al., 1997). (4) Monoclonal anti-FcgRII antibodies (IV.3) are purified from the ascites fluid of mice inoculated with hybridoma HB 217, which is obtained from the American Type Culture Collection (Rockville, MD, USA). F(abV)2 fragments of the IV.3 antibody are prepared essentially as described in the Pierce catalog (Rockford, IL, USA). Briefly, the antibodies are digested with pepsin and intact antibodies are eliminated by adding protein A and protein G beads. The integrity and purity of the F(abV)2 fragments are verified by their ability to label intact human neutrophils as determined by flow cytometry as well as by their neutrophil-activating properties (calcium mobilization, superoxide generation, tyrosine phosphorylation). 4.6. Reagents Soybean trypsin inhibitor, cholera toxin B-HRP and sodium orthovanadate (Na3VO4) are purchased from Sigma-Aldrich Canada. Di-isopropylfluorophosphate (DFP) (NP-40 10% and 100% solution) are obtained from Calbiochem. p-Nitrophenylphosphate, aprotinin and leupeptin are purchased from ICN Pharmaceuticals (Costa Mesa, CA, USA). Sephadex G-10, protein A sepharose are purchased from Pharmacia Biotech. SAM68 – GST fusion protein is obtained from Santa Cruz Biotechnology. 4.7. Special equipment 1. 2. 3. 4. 5. 6. 7. 8. 9.

Centrifuge Microcentrifuge Thermomixer R at 37 and 30 jC (Eppendorf) Dry bath at 100 jC Sonicator Rotator platform at 4 jC Peristaltic pump Electrophoresis slab gel apparatus Electrophoretic Transfer Unit

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10. Power supply 11. Rocking platform 12. Microcentrifuge tubes.

5. Detailed procedures 5.1. Cell preparation Peripheral blood is obtained from healthy adult volunteers and collected on isocitrate anticoagulant. Neutrophil suspensions are prepared sterilely as previously described (Pouliot et al., 1998), except that sedimentation of erythrocytes is performed using 2% dextran solution diluted in HBSS. The cells are resuspended at a concentration of 107 cells/ml in HBSS, pH 7.4, containing 1.6 mM calcium but no magnesium in order to minimize aggregation. Cell preparations with non spherical morphologies (an indication of pre-activation) are excluded from the study. 5.2. Cell stimulation Before all stimulations, neutrophil suspensions (4  107 cells/ml) are pre-incubated at room temperature with 1 mM DFP for 5 min. Neutrophils suspensions of the same concentration are then stimulated with the desired agonists at 37 jC. To cross-link CD32, the cell suspensions are incubated with 2.0 Ag/ml of anti-FcgRII (IV.3) antibodies for 1 min at 37 jC and then with 20 Ag/ml of F(abV)2 goat anti-mouse Fc antibody (Jackson Immune Research) for 1 min at 37 jC or the indicated periods of time. For immunoprecipitations, the F(abV)2 fragment of IV.3 is used instead of the whole antibody. 5.3. Preparation of total cell lysates under denaturing conditions After stimulation, the reactions are stopped by transferring 100 Al of the cell suspensions directly to an equal volume of boiling 2  sample buffer. The samples are boiled for 7 min and vortexed frequently to ensure that the cells have dissolved properly. The samples are loaded immediately on SDS polyacrylamide gels, or stored at 80 jC (if frozen, it is best to reheat samples before loading onto gels.). It is critical

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to add the cells directly to boiling sample buffer as this greatly reduces the background levels of tyrosine phosphorylation due to nonspecific activation. The addition of DFP (1 –4 mM) to the resuspension buffer helps minimize proteolysis and activation caused by centrifugation (Gilbert et al., 2001). The conditions outlined above should apply equally to other leukocyte populations. 5.4. Hypotonic cell lysis After stimulation, the reactions are rapidly stopped by transferring the cell suspensions to precooled ( 20 jC) 1.5 ml microcentrifuge tubes. The cells are then centrifuged for 5 –10 s at 6000  g in a microcentrifuge and the pellets resuspended at a final concentration of 4  107 cells/ml in the hypotonic lysis buffer (HLB). For better inhibition of phosphatases, 10 mM p-nitrophenylphosphate may be added to the lysis buffer. After a 5-min incubation at 4 jC, the lysates are centrifuged at 600  g for 10 min at 4 jC. Supernatants generated can be used either for immunoprecipitation under native or denaturing conditions, or for the determination of tyrosine kinase activities, or transferred to an equal volume of boiling 2  SB. Similarly, the resulting pellets are either resuspended in HLB and then transferred to an equal volume of boiling 2  SB, or ILB or HyperLB for immunoprecipitation or determination of tyrosine kinase activity. Nuclear integrity and total cell lysis are routinely verified by light microscopy. 5.5. Isotonic cell lysis After stimulation, the reactions are rapidly stopped by transferring the cell suspensions into precooled ( 20 jC) 1.5 ml microcentrifuge tubes. The cell suspensions are then centrifuged for 5 – 10 s at 6000  g in a microcentrifuge. The supernatants are removed and the cell pellets are resuspended and incubated for 5 min at 4  107 cells/ml at 4 jC in cold isotonic lysis buffer (ILB). The lysates are centrifuged at 600  g for 10 min at 4 jC. Aliquots of the supernatants are added to an equal volume of boiling 2  SB. The pellets are resuspended in ILB and then transferred to an equal volume of boiling 2  SB.

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5.6. Sequential solubilization The soluble and insoluble fractions of the hypotonic lysis are individually processed for subsequent analysis. The soluble fraction is centrifuged at 180,000  g at 4 jC for 45 min. The supernatant is analyzed by transfer of an aliquot into 2  SB. The pellet is dissolved in 1 volume of HLB and transferred to boiling 2  SB. The fraction insoluble in HLB is resuspended in ILB and centrifuged for 10 min at 13,000  g. The soluble material of this second lysis is transferred into 2  SB and the pellet is resuspended in the hypertonic lysis buffer (HyperLB). Two 5-s sonication steps may be necessary for maximal pellet extraction depending on the individual sonicator used. Some adjustment may be necessary to maximize extraction and preservation of intact phosphorylated products. The lysates are centrifuged at 13,000  g for 10 min at 4 jC. Aliquots of the supernatants of each fraction (soluble HLB to soluble HyperLB) are transferred to an equal volume of boiling 2  SB. The pellets are resuspended in HLB and then diluted in the same volume of 2  SB. The samples are then electrophoresed as described above. The soluble fractions of HyperLB are also used under native conditions for immunoprecipitation and tyrosine kinase activity. These fractions (soluble HyperLB) can also be obtained by direct addition of HyperLB to the HLB pellets. The experimental scheme illustrated in Fig. 1 shows the details of this procedure. 5.7. Immunoprecipitation under denaturing conditions Neutrophils are incubated and stimulated as described above. Aliquots (500 Al) of the cells are lysed by direct transfer to an equal volume of boiling 2  lysis buffer (2  LB) and boiled for 7 min. Immunoprecipitations are performed as previously described (Al-Shami et al., 1997b). Briefly, lysates are filtered through sephadex G-10 columns to remove the denaturing agents. The filtered lysates are precleared with protein A-sepharose at 4 jC for 30 min in the presence of 1% NP-40, 2 mM orthovanadate, 10 Ag/ml leupeptin and 10 Ag/ml aprotinin. The samples are then immunoprecipitated using 2 Ag of anti-Cbl, 2 Ag of anti-Syk or 6 Ag of anti-CD32 previously bound to protein A-sepharose for 90 min at

4 jC on a rotator platform with constant end-over-end mixing. The beads are collected and washed four times with a lysis buffer containing 137 mM NaCl, 1% NP-40 but no SDS, h-mercaptoethanol or bromophenol blue. Sample buffer (40 Al, 2  ) is added to the beads which are then boiled for 7 min. The proteins in the samples are then separated by electrophoresis as described above. The membranes are blotted with anti-phosphotyrosine or with the immunoprecipitating antibodies (anti-Syk, anti-Cbl or antiCD32) for visualizing the amounts of precipitated protein. 5.8. Native immunoprecipitation and tyrosine kinase activity The aliquots of HLB or HyperLB supernatant lysates are mixed with HyperLB or HLB, respectively, to bring back the NaCl and NP-40 concentrations to 137 mM and 1%, respectively. These isotonic lysates are immunoprecipitated using 1.5 Ag of anti-Lyn or 3 Ag of rabbit anti-mouse heavy/light chain antibodies (for CD32 immunoprecipitation) for 90 min at 4 jC on a rotator platform with constant end-over-end mixing. The RAM antibodies recognize the antiCD32 fragment antibodies fixed on the cells during stimulation and do not react with the goat anti-mouse antibodies used for cross-linking CD32. Fifty microliters (30% slurry) of protein A-sepharose is then added and the samples are incubated for 1 h at 4 jC. The beads are collected and washed four times with ILB buffer without EDTA. The beads are incubated at 4 jC in kinase buffer and transferred to 37 jC for the indicated times. The reactions are stopped by a quick spin and the supernatants are discarded and then 2  SB is added to the beads. In the case of SAM68– GST, when present in the KB mix, the supernatants are precipitated with sepharose – GST beads and washed twice with ILB buffer. Sample buffer (40 Al, 2  ) is added to the beads which are then boiled for 7 min. The proteins in the samples are then separated by electrophoresis as described above. The membranes are blotted with the anti-phosphotyrosine or with the specific immunoprecipitation antibodies for visualizing the amounts of precipitated protein. Immunoprecipitation with control rabbit IgG followed by kinase activity is undertaken in parallel to verify the specificity of the precipitations.

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5.9. Detailed electrophoresis and immunoblotting procedure Prior to loading onto 7.5– 20% SDS-PAGE acylamide gels, samples in 2  SB are boiled for 7 min. Proteins are then transferred to Immobilon PVDF membranes (Millipore, Bedford, MA, USA). Gradient gels (7.5 –20%) and Immobilon PVDF membranes have been found to be optimal for monitoring the overall profile of tyrosine phosphorylation and for effectively binding the majority of transferred proteins. After transfer, the proteins are visualized with a 1% (w/v) Ponceau solution and the molecular weights markers are identified. Nonspecific sites are blocked with a 2% (w/v) gelatin solution for 30 – 60 min on a rocking platform (not an orbital shaker). Dried milk (i.e., Blotto) is not recommended as a blocking solution for tyrosine phosphorylation immunoblotting as it increases background. All subsequent incubations are performed on a rocking platform with constant but not too vigorous rocking. We have found it important to have only one membrane per container. The blocking solution is discarded and replaced with a 2% gelatin solution containing a 1:4000 dilution of anti-phosphotyrosine antibody (UBI 05 321) and incubated for 1 h at 37 jC. The containers are rinsed with water to remove any residual antibody solution and the membranes are then washed with TBS 1  (three washes of 2 min each). The membranes are then incubated with conjugated secondary antibody, either goat anti-mouse-HRP antibodies ((#NXL-931) 1:20,000, Amersham/Pharmacia, Baie D’Urfe´, QC, Canada) or donkey anti-mouse-HRP antibodies ([#715-035-150] Jackson Immune Research) in 2% gelatin solution or in TBS/Tween for 30 –60 min at 37 jC or at room temperature equivalently. The secondary antibody solution can only be used once and should be made fresh for each assay. The containers are rinsed with water and the membranes washed four times for 5 min in a container with a large volume of TBS/Tween (e.g., for a 12  12 cm membrane, around 125 ml of TBS/Tween per wash). Care needs to be taken when handling the membranes as any damage can increase background staining. The detection system solution is prepared as described in the data sheets of the manufacturer (NEN Life Science, Boston, MA). The membranes are placed protein facedown in a clean container with the detection solution for 1 min

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at room temperature without agitation. The solution is dripped away for a few seconds and the membranes are placed on a sheet of plastic food wrap large enough to cover the membrane completely. The edges are sealed and the membranes placed protein side up in an autoradiography cassette. For clear and wellfocussed bands, the exposure of the film to the membranes in the cassette should be conducted without intensifying screens. For the other antibodies, immunoblotting is performed using the appropriate antibodies (anti-Cbl (1:1000), anti-CD32 (1:1000), anti-SAM68 (1:1000) and anti-Syk (1:1000)) and revealed using the Renaissance detection system (NEN Life Science) using HRP-conjugated secondary donkey anti-mouse [#715-035-150] or donkey antirabbit [#711-035-152] antibodies (Jackson Immune Research) at a dilution of 1:20,000 as described above.

6. Results 6.1. Experimental schema of the sequential solubilization protocol Tyrosine phosphorylation in total neutrophil lysates can be analyzed by immunoprecipitation under denaturing conditions as illustrated in the left side of Fig. 1. A major limitation of this technique (Al-Shami et al., 1997b) is that it is impossible to simultaneously monitor tyrosine kinase activity. Aware of this limitation, we developed an alternate protocol which preserved the tyrosine phosphorylation and the kinase activity and which is based on an initial lysis in a hypotonic buffer (Gilbert et al., 2002). The soluble and insoluble fractions of the hypotonic lysis are therefore individually processed for subsequent analysis as indicated in the right side of Fig. 1. We wanted to better characterize the soluble fraction of the hypotonic lysis. After stimulation, the soluble fraction of the hypotonic lysis is centrifuged for 45 min at 180,000  g. The resulting soluble and insoluble fractions can be analyzed by immunoblotting with anti-phosphotyrosine or other antibodies. Since most of the tyrosine-phosphorylated proteins are recovered in the pellets following hypo-

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Fig. 1. Experimental schema of sequential solubilization protocol. This schema represents several alternatives for studying the tyrosine kinase pathway in the neutrophil. This procedure is also applicable for the other cells.

tonic lysis, a further extraction of this pellet is warranted in order to carry out immunoprecipitations from this fraction. The HLB-insoluble fraction is sequentially lysed in buffers of increasing tonicity, the composition of which is described in Section 4 (detailed procedures). Briefly, the HLBinsoluble material is resuspended in ILB (dotted line) or in HyperLB, and separated after centrifugation into the soluble and insoluble fractions. The ILB-insoluble material is then resuspended in HyperLB, and soluble and insoluble fractions are isolated after centrifugation. The relevant fractions are analyzed by immunoblotting with anti-phosphotyrosine antibodies (Fig. 2, panel A), or immunoprecipitated for the analysis of the translocation, phosphorylation or kinase activity of various proteins as illustrated in Fig. 3.

6.2. Effect of tonicity of the lysis buffer on the preservation and distribution of tyrosine phosphorylation patterns and signaling-associated molecules The difficulties associated with the preservation of phosphotyrosine profiles and protein integrity in human neutrophils have previously been described (Al-Shami et al., 1997b; Naccache et al., 1997). The extraction of tyrosine-phosphorylated proteins is known to be affected by the composition of the lysis buffer (Ignatoski and Verderame, 1996); the pH, presence of phosphate, salt concentration, cell concentration, presence of cations, protease and phosphatase inhibitors can all modify the detergent behavior and the solubilization of individual proteins. Previous studies have reported the solubilization of proteins without affecting the structure of the

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Fig. 2. Distribution and preservation of tyrosine phosphorylation profiles in HLB and ILB. Neutrophils (4  107 cells/ml) are stimulated by cross-linking CD32 as indicated in the detailed procedure. The reactions are stopped by a transfer of cell aliquots into a precooled Eppendorf tube followed by rapid centrifugation. The appropriate lysis buffer is then added to the cell pellet, HLB for lanes 3 – 6 and ILB for lanes 7 – 10 or in the boiling 2  SB for lanes 1 and 2. Aliquots of soluble (lanes 3,4 and 7,8) and insoluble (lanes 5,6 and 9,10) fractions are diluted in an equal volume of 2  SB. The samples are analysed by Coomassie blue protein staining (panel A) or by Western blot with anti-phosphotyrosine (panel B).

nuclei using a hypotonic lysis buffer coupled with low concentrations of detergents (0.1% NP-40) (Pouliot et al., 1996; McDonald et al., 1998; Gilbert et al., 2001, 2002). The data in Fig. 2 compare this hypotonic buffer (HLB) with a more classical isotonic RIPA buffer (ILB). The soluble and insoluble fractions of the two lysis buffers (lanes 3 – 10) are compared to the whole cell lysates (lanes 1– 2). The

protein (Coomassie blue staining) and tyrosine phosphorylation profiles are illustrated in panels A and B for the soluble (lanes 3– 4, 7– 8) and insoluble (lanes 5– 6, 9– 10) fractions derived from these cell lysis protocols. To study the tyrosine phosphorylation profile, neutrophils are stimulated by cross-linking CD32 for 1 min at 37 jC as described in Section 4 (detailed procedure). The samples are analyzed by

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Fig. 3. Tyrosine kinase activity in the hypotonic and hypertonic soluble fractions. Neutrophils (4  107 cells/ml) are stimulated by cross-linking CD32 for 1 min at 37 jC as described in detailed procedure. The reactions are stopped by a transfer in a precooled Eppendorf tube followed by rapid centrifugation. The HLB + p-nitrophenylphosphate is added to the cell pellet. The aliquots (200 Al or 8  106 equivalent cells) of the HLB lysate supernatants are diluted with 200 Al of HyperLB and 200 Al of HLB. The pellet of the hypotonic lysis is directly incubated in a second lysis buffer (HyperLB) and sonicated 2  5 s. The soluble material (200 Al or 8  106 equivalent cells) of this lysate are diluted in 2 volumes of HLB and incubated with the anti-Lyn antibodies, and the kinase activity is performed as described in the detailed procedure. Aliquots of each fraction are analysed by immunoblotting with an anti-phosphotyrosine antibodies in a panel A. Autophosphorylation is visualised by Western blot with anti-phosphotyrosine antibody in a panel B.

Coomassie blue protein staining (panel A) or by immunoblotting with anti-phosphotyrosine antibodies (panel B), respectively.

The percentage of lysed cells and the integrity of the nuclei are routinely verified by light microscopy (data not shown) or by immunoblot analysis with

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nuclear markers such as p62 nucleoporin (a constituent of nuclear membranes) (Fig. 4). This confirmed that the cells are completely lysed in the two buffers and that nuclear integrity is better preserved in HLB than in ILB (data not shown). Coomassie blue staining (panel A) of these samples indicated that the amount of protein extracted with the two lysis buffers is roughly similar. The tyrosine phosphorylation profile induced by CD32 cross-linking, as monitored after the direct transfer of the cells into sample buffer is illustrated in the first two lanes of panel B and served as a reference point for the evaluation of the adequacy of the native cell lysates. Two major conclusions can be drawn from monitoring the tyrosine phosphorylation profiles in the various fractions (panel B). Firstly, tyrosine-phosphorylated proteins are concentrated in the insoluble fractions in both lysis protocols (lanes 5 –6 and 9 –10). Secondly, the tyrosine phosphorylation profiles are significantly better preserved in HLB than in ILB (lanes 4– 6 versus lanes 8 –10). Based on the three criteria, (i) prevention of proteolysis (Gilbert et al., 2002), (ii) preservation of tyrosine phosphorylation and (iii) day-to-day reproducibility of tyrosine phosphorylation levels (data not

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shown), the data in Fig. 2 indicate that HLB is a superior buffer for the preparation of starting material from which to study tyrosine phosphorylationdependent signaling events in human neutrophils than is ILB. 6.3. Sequential solubilization of the tyrosine-phosphorylated proteins Since most of the tyrosine-phosphorylated proteins are recovered in the pellets of the hypotonic lysis, a further extraction of this pellet is warranted in order to be able to carry out immunoprecipitation from this fraction. On the other hand, it is important to better characterize the soluble fraction of the hypotonic lysis. The soluble and insoluble fractions of the hypotonic lysis buffer are therefore individually processed for subsequent analysis. Fig. 3 shows the anti-phosphotyrosine analysis of the soluble and the insoluble fractions of the HLB (panel A) coupled with the evaluation of the kinase activity of Lyn (panel B). After stimulation with the IV.3 F(abV)2, the soluble fractions (lanes 3 – 4) of the hypotonic lysis are conserved for immunoprecipitation with anti-Lyn antibodies. The HLB-insoluble fraction is directly lysed in HyperLB buffers and the soluble fractions of this second lysis are conserved. The relevant fractions are analyzed by immunoblotting with antiphosphotyrosine antibodies (panel A). The whole cell lysates (lanes 1– 2) served as the reference point for the extent of stimulation as far as the global tyrosine phosphorylation profile is concerned. As shown in Fig. 3, the majority of the tyrosine-phosphorylated proteins present in the pellet of the HLB are solubilized and preserved in HyperLB (lanes 7– 8) although some tyrosine-phosphorylated proteins (lanes 9 –10) remained insoluble. 6.4. Lyn activity following its extraction from HyperLB

Fig. 4. Fraction characterization. Neutrophils (4  107 cells/ml) are lysed in 2  SB or in HLB buffer. The insoluble materials of the HLB lysis are further processed in HyperLB buffer as described in the detailed procedure. Aliquots of each fraction are diluted in 2  SB and analysed by Western blot with specific antibodies or Cholera toxin HRP-coupled.

In order to assess the state of the proteins extracted in HyperLB, the immunoreactivity and enzymatic activity of the Src kinase Lyn are tested next in the soluble fractions of the HLB and HyperLB lysates. It has previously been shown that Lyn is activated in response to MSU crystals (Gau-

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dry et al., 1995; Gilbert et al., 2002) and the involvement of Lyn in activation via FcgRII has also been described (Marcilla et al., 1995; Ibarrola et al., 1997). To confirm this result following the sequential lysis described here, neutrophils are stimulated by pre-incubation with IV.3 F(abV)2 fragments followed by addition of GAM for 1 min before being lysed in HLB. HLB-soluble and -insoluble fractions are prepared as described above. As the HLB-insoluble material is also insoluble in ILB, the HLBinsoluble fractions are directly resuspended in HyperLB. Soluble and insoluble fractions are prepared and Lyn is immunoprecipitated from the HLBand HyperLB-soluble fractions. The results shown in Fig. 3B demonstrate that Lyn immunoprecipitated from the HyperLB-soluble fractions retains in vitro kinase activity. Furthermore, a stimulatory effect of CD32 ligation on the auto-kinase activity of Lyn is only detectable in the HyperLB lysates (Fig. 3B, right side). Reblots indicate that equal amounts of Lyn are immunoprecipitated and loaded in the control and CD32-stimulated cells (data not shown). 6.5. Characterization of the fractions A partial characterization of the fractions obtained during the sequential lysis protocol followed herein is then carried out. Cytosolic markers (myeloid-related protein-14 (MRP-14) and lactate dehydrogenase (LDH)) are found predominantly in the soluble fraction of the first lysis with HLB and in the soluble fractions of the ultracentrifugation steps. Of relevance, the receptor for cholera toxin (GM1) (Hart, 1975), a major constituent of lipid rafts, is totally insoluble in HLB buffer but is solubilized in HyperLB (Fig. 4). Early endosome-associated protein-1 (EEA-1), an endosomal marker, is equally distributed between the soluble and insoluble fractions of HLB. The HyperLB step completely solubilized EEA-1. Lysosome-associated membrane protein-1 (LAMP-1) is entirely recovered in the HLB soluble fraction. Cytoskeletal markers are also analyzed. A significant translocation of actin to the insoluble fraction is evident after CD32 cross-linking as monitored by Coomassie blue staining (Fig. 2). Paxillin and ezrin are equally distributed between the soluble and the insoluble fractions of the hypotonic lysis (Fig. 4). Extraction with the hypertonic lysis buffer solubilized these

proteins. Finally, nuclear markers (p62 nucleoporin) remain insoluble and are found in the pellets of the HyperLB step (Fig. 4). 6.6. Association of kinase activity and Syk with CD32 precipitated in the HyperLB fractions Stimulation of CD32 by cross-linking led to its partitioning in the insoluble fraction in non-ionic detergents, and, subsequently, its degradation (the latter being reduced in the presence of tyrosine kinase inhibitors (Barabe´ et al., 2002)). To confirm the association of tyrosine kinase with CD32, CD32 is cross-linked for increasing periods of time, the cell pellets are lysed in HLB buffer and the insoluble fractions are submitted to a second lysis in the HyperLB as described above. The soluble material of HyperLB fraction is incubated with 3 Ag of rabbit anti mouse heavy/light chain antibodies and processed for immunoprecipitation of CD32. Control experiments established that the anti-CD32 antibodies fixed on the cell by stimulation before lysis are preserved in the presence of high salt concentrations and following the sonication step. The immunoprecipitates are incubated in a kinase buffer containing ATP and GST – SAM68 fusion protein (as an exogenous substrate) for 10 min at 37 jC. The GST-fusion protein are recovered with sepharose –GST beads and the CD32 immunoprecipitates are denaturated in 2  SB before analysis by immunoblots (details in Section 4). Immunoblots with an anti-phosphotyrosine antibody (Fig. 5, panel A) show a time-dependent association of tyrosine-phosphorylated proteins with the CD32 immunoprecipitates. Two prominent bands with migration characteristics that correspond to those of CD32 and of the tyrosine Syk are consistently detected. It should be pointed out that Src kinases are masked by interference by the heavy chains of the immunoprecipitating RAM antibodies. Association of tyrosine kinase Syk with ITAM receptor has been described upon CD32, CD64 and TCR activation (Marcilla et al., 1995; Daeron, 1997; Bonnerot et al., 1998). Immunomunoblot with anti-Syk antibodies (panel B) reveal that the tyrosine kinase Syk is present in the major tyrosine-phosphorylated band at 66 kDa observed in a panel A, and that this association increased after CD32 ligation in a time-dependent manner. Immunoblot with the anti-CD32 in panel

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GST – SAM68 precipitated with the anti-phosphotyrosine antibodies revealed that the CD32-associated kinases are able to phosphorylate SAM68 – GST within 1 min of ligation, and that this phosphorylation

Fig. 5. Association of kinase Activity and Syk with the CD32 precipitated in the HyperLB fractions. Neutrophils (4  107 cells/ ml) are stimulated by cross-linking CD32 for the indicated time at 37 jC as described in the detailed procedure. The reactions are stopped by a transfer in a precooled Eppendorf tube followed by rapid centrifugation. The cells are lysed with the HLB. The pellet of the hypotonic lysis is directly incubated in a second lysis buffer (HyperLB) and sonicated 2  5 s. The soluble material (400 Al or 16  106 equivalent cells) of this lysis are diluted in 2 volumes of HLB and incubated with 3 Ag of rabbit anti-mouse heavy/light chain antibodies, and the immunoprecipitation and kinase activity assays are performed as described in the detailed procedure. Kinase activity associated with the CD32 is visualized by immunoblot with an antiphosphotyrosine antibodies (panel A). Protein associated with the CD32 is identified in panel B by immunoblotting with an anti-Syk antibodies. Amount of CD32 immunoprecipitated is evaluated in a panel C with an anti-CD32 antibodies. Activity of associated tyrosine kinases is verified in SAM68 – GST precipitates as shown in panels D and E by immunoblot with an anti-phosphotyrosine antibodies (panel D) and anti-SAM68 (panel E).

C shows that the amount of immunoprecipitated CD32 is approximately equal. The results in panels D and E confirm the presence of active tyrosine kinase(s)-associated with CD32. Immunoblot of

Fig. 6. Immunoprecipitation of tyrosine-phosphorylated proteins from the HLB soluble and insoluble fractions. Neutrophils (4  107 cells/ml) are stimulated by cross-linking CD32 as indicated in Section 4. The reactions are stopped by a transfer of cell aliquots in a precooled Eppendorf tube followed by rapid centrifugation. The cell pellets are resuspended in HLB and the soluble and insoluble fractions are diluted by 1/2 in boiling 2  LB. Immunoprecipitations under denaturing conditions are performed as described in the detailed procedure with the anti-Cbl, anti-Syk or anti-C32 antibodies. Phosphorylated proteins are revealed with an antiphosphotyrosine antibody (upper panels), and the amount of immunoprecipitated protein is visualized by immunoblot with the respective antibodies (lower panels).

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is maintained for least 10 min. Immunoblot with the SAM68 antibodies revealed that equal amounts of GST – SAM68 are precipitated and loaded onto each lane. 6.7. Tyrosine-phosphorylated proteins are concentrated in the insoluble fractions The results described above revealed a heterogenous distribution of phosphorylated proteins among the soluble and insoluble fractions derived from the lysis protocols after CD32 engagement. We therefore monitored more closely three major tyrosine-phosphorylated substrates previously identified following CD32 cross-linking, namely, Cbl, Syk and CD32 itself (Rollet-Labelle et al., 2000) (Fig. 6). Briefly, aliquots of the soluble fractions derived from the lysis of control and CD32-stimulated cells in HLB are resuspended in an equal volume of boiling 2  LB as described in above. The insoluble material is resuspended in the same volume of HLB as the soluble fractions and subsequently in an equal volume of boiling 2  LB. Cbl, Syk and CD32 are immunoprecipitated from both fractions. The precipitates are probed with antiphosphotyrosine antibodies as well as with anti-Cbl, anti-Syk or anti-CD32 antibodies (panels A, B and C, respectively). The results obtained show that phosphorylated Cbl, Syk and CD32 are highly concentrated in the insoluble fractions in agreement with the observations of Figs. 3 and 5. Reblots with anti-Cbl, anti-Syk or anti-CD32 antibodies (lower panels) indicate that cross-linking of CD32 did not grossly modulate the amounts of Cbl and Syk present in each fraction. On the other hand, a significant decrease in the level of CD32 is observed in the soluble fractions, and this is compensated for, in part at least, by an increase in the level of CD32 in the insoluble fractions.

7. Discussion The protocols described above provide a framework for the immunobiochemical investigation of tyrosine phosphorylation-dependent signaling pathways in human neutrophils. The basis of this method is an initial lysis in a hypotonic buffer. Under these conditions, the overall tyrosine phosphorylation pro-

file is preserved to a major extent. The two fractions thus obtained (soluble and insoluble) can then be further analyzed, either directly (in the case of the soluble fractions) or following solubilization in buffers of increasing tonicities (in the case of the original insoluble fractions). Each fraction can be analyzed by immunoblotting or by monitoring enzymatic activities. A major characteristic of these protocols is that they allow retention of the tyrosine phosphorylation profile, maintain enzymatic activities and preserve associations with surface receptors. This procedure also established that the detergent solubility of individual tyrosine-phosphorylated substrates differed, not only according to the protein under investigation, but also in response to the specific agonist used (Gilbert et al., 2002). The severe experimental problems associated with the preparation of neutrophil lysates (proteolysis, dephosphorylation or hyperphosphorylation) are widely acknowledged but poorly documented. A previous attempt to preserve the original patterns of tyrosine phosphorylation relied on a denaturing lysis protocol which, while effective, eliminated the possibility of studying protein – protein interactions and enzymatic activities (i.e., kinases and phosphatases) in the lysates (Al-Shami et al., 1997b). The sequential lysis protocol described in the present manuscript overcomes these limitations in that the lysates prepared under native conditions maintained to a significant extent their profile of tyrosine phosphorylation (Fig. 2) as well as their enzymatic activities (Figs. 3– 5) and protein – protein associations with surface receptor (Fig. 5). Furthermore, whereas the stimulation of tyrosine phosphorylation is transient in intact neutrophils, we observed that the tyrosine phosphorylation in the insoluble fractions was maintained for up to 60 min following cell lysis. This can be explained by the technique used to stop the stimulation, i.e., transfer of the cells in precooled tubes followed by rapid centrifugation. This approach immediately eliminates the incubation medium which may contain degradative enzymes (proteases) and phosphatases released by the cells during stimulation. One of the main findings of the present investigation was that the detergent extractability of the tyrosine-phosphorylated substrates was both substrate- and agonist-dependent (Gilbert et al., 2002). Nevertheless,

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a large percentage of the tyrosine-phosphorylated proteins was insoluble in regular RIPA buffers of hypo- or iso-tonicity (Fig. 2). This point raises questions about the interpretation of immunoprecipitation studies in which the fraction that is soluble in these lysis buffers is used as starting material in the absence of a detailed picture of the distribution of the protein being examined. Furthermore, the level of phosphorylation of various proteins can be artifactually increased by the presence of Mg2 + in the lysis buffers (Gilbert et al., 2002), a procedure sometimes adopted to help preserve membrane and cytoskeletal integrity. Once again, this may impact on the interpretation of the functional significance of apparent stimulation, or lack of stimulation, of the tyrosine phosphorylation of specific substrates in those cases where the profiles of tyrosine phosphorylation of the lysates used as starting material for the immunoprecipitation differs significantly from that of whole cells. The importance of knowing the distribution of proteins in detergent extracts of the cells is strikingly illustrated by the behavior of CD32, Cbl and Syk in response to stimulation by CD32 ligation. All three proteins were rapidly tyrosine-phosphorylated following CD32 cross-linking (Marcilla et al., 1995; Naccache et al., 1997; Rollet-Labelle et al., 2000) and, as shown in Fig. 6, they were recovered in both the soluble and the insoluble fractions. However, in all three cases, the tyrosine-phosphorylated fractions of these proteins were highly concentrated in the detergent-insoluble fractions. The physiological relevance of the soluble form of these proteins is therefore likely to differ from that present in the insoluble fraction. Cross-linking of CD32 induces its insolubility in nonionic detergents, thereby implicating a potential role for detergent-resistant membranes (Barabe´ et al., 2002; Gilbert et al., 2002). Insolubility of membrane receptors coupled to kinase activities were also observed in T cell lines (Solomon et al., 1998) and in various others cells (Zhou et al., 1995). In the case of the stimulation of CD32, we were able to detect not only its insolubility (Barabe´ et al., 2002; Gilbert et al., 2002) (Figs. 5 and 6) but also the appearance of tyrosine kinase activity that rapidly (after 1 min of ligation) associated with CD32 in the insoluble fraction of the HLB. Association of Syk with ITAM motifs after tyrosine phosphorylation by Src family kinase is a central feature of the classical models for

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ITAM-motif activation. The results obtained in Fig. 5 show that our procedures allowed a time-dependent association of Syk with CD32 to be detected (panel B). We also observed the tyrosine phosphorylation of a prominent band with electrophoretic migration characteristics of Syk when we incubated the precipitated cross-linked CD32 in a kinase buffer (panel A). Pretreatment of the cells with the Src inhibitor PP-2 abolished this association (data not shown) in accordance with the classical model of ITAM activation described by others (Marcilla et al., 1995; Daeron, 1997; Bonnerot et al., 1998). The functional relevance of the above considerations is also highlighted by the results of the experiments in which the kinase activity of Lyn was monitored in the soluble fractions of both the hypotonic and hypertonic lysis buffers (Fig. 3). The enzyme remained active in both fractions, an indication of preservation of structure and function in the lysates. Of more functional relevance, however, was the observation that the stimulatory effect of CD32 cross-linking on the activity of Lyn was only readily detectable in the hypertonic buffer lysate. A study of the soluble fraction of the hypotonic lysate would have missed this effect, while a lysis in an isotonic buffer would be likely to result in a significant level of signal distortion (proteolysis, dephosphorylation). The sequential lysis protocol characterized in the present study overcomes several of these problems by preserving the original phosphorylation status and by sequentially giving access to the various fractions. This observation indicates that both fractions must always be examined in these kinds of studies. These data are consistent with the results of Zhou et al. (1995), who previously reported a partitioning of Src kinase activities between in detergent insoluble fractions derived from adherent neutrophils. These enzymes are involved in the early steps of several neutrophil responses and are differentially regulated depending of their intracellular distribution (Welch and Maridonneau-Parini, 1997). Our results also indicate that, depending on the agonist used, the distribution of the tyrosine-phosphorylated substrates varies and must be individually characterized. Furthermore, the addition of cofactors in the lysis buffer must be carefully controlled. In conclusion, we have demonstrated that the detergent-insoluble fractions cannot be excluded in

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biochemical studies of stimulated human neutrophils. The concentration of signaling molecules in these fractions may explain some of the controversial results concerning tyrosine-phosphorylated proteins, tyrosine kinase activities or protein associations that have been reported in stimulated neutrophils. The present approach, which is based on the examination of all cell fractions, may help to understand the biochemical mechanisms regulating the functional responsiveness of neutrophils. This method may also be extended to the monitoring of phosphatase activities if Na3VO4 is omitted from the lysis buffers allowing the evaluation of the balance of kinase and phosphatase activities in stimulated cells, which is most likely to be a key to the regulation of several functional responses in many cells.

leukocytes. Implications for immune complex activation of the respiratory burst. J. Biol. Chem. 270, 13553 – 13560.

Acknowledgements Supported in part by grants from the Canadian Institutes of Health Research. C. Gilbert is supported by fellowships from the K.M Hunter Charitable Foundation, the Canadian Institutes of Health Research and the Fonds pour la Formation de Chercheurs et l’Aide a` la Recherche and the Fonds de la Recherche en Sante´ du Que´bec.

References 8. Essential literature references Al-Shami, A., Gilbert, C., Barabe´, F., Gaudry, M., Naccache, P.H., 1997b. Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. J. Immunol. Methods 202, 183– 191. Gilbert, C., Rollet-Labelle, E., Naccache, P.H., 2002. Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates: II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling. J. Immunol. Methods 261, 85– 101. Ignatoski, K.M., Verderame, M.F., 1996. Lysis buffer composition dramatically affects extraction of phosphotyrosine-containing proteins. BioTechniques 20, 794 – 796. Welch, H., Maridonneau-Parini, I., 1997. Lyn and Fgr are activated in distinct membrane fractions of human granulocytic cells. Oncogene 15, 2021 –2029. Yan, S.R., Fumagalli, L., Berton, G., 1996. Activation of SRC family kinases in human neutrophils. Evidence that p58C-FGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity. FEBS Lett. 380, 198– 203. Zhou, M.J., Lublin, D.M., Link, D.C., Brown, E.J., 1995. Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear

Al-Shami, A., Bourgoin, S.G., Naccache, P.H., 1997a. Granulocyte – macrophage colony-stimulating factor-activated signaling pathways in human neutrophils: I. Tyrosine phosphorylationdependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters. Blood 89, 1035 – 1044. Al-Shami, A., Gilbert, C., Barabe, F., Gaudry, M., Naccache, P.H., 1997b. Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. J. Immunol. Methods 202, 183 – 191. Barabe´, F., Gilbert, C., Liao, N., Bourgoin, S.G., Naccache, P.H., 1998. Crystal-induced neutrophil activation: VI. Involvement of FcgammaRIIIB (CD16) and CD11b in response to inflammatory microcrystals. FASEB J. 12, 209 – 220. Barabe´, F., Rollet-Labelle, E., Gilbert, C., Fernandes, M.J., Naccache, S.N., Naccache, P.H., 2002. Early events in the activation of FcgammaRIIA in human neutrophils: stimulated insolubilization, translocation to detergent-resistant domains, and degradation of FcgammaRIIA. J. Immunol. 168, 4042 – 4049. Bonnerot, C., Briken, V., Brachet, V., Lankar, D., Cassard, S., Jabri, B., Amigorena, S., 1998. Syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes. EMBO J. 17, 4606 – 4616. Brown, D.A., London, E., 1998. Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 14, 111 – 136. Daeron, M., 1997. Fc receptor biology. Annu. Rev. Immunol. 15, 203 – 234. Gaudry, M., Gilbert, C., Barabe´, F., Poubelle, P.E., Naccache, P.H., 1995. Activation of Lyn is a common element of the stimulation of human neutrophils by soluble and particulate agonists. Blood 86, 3567 – 3574. Gilbert, C., Barabe´, F., Rollet-Labelle, E., Bourgoin, S.G., McColl, S.R., Damaj, B.B., Naccache, P.H., 2001. Evidence for a role for SAM68 in the responses of human neutrophils to ligation of CD32 and to monosodium urate crystals. J. Immunol. 166, 4664 – 4671. Gilbert, C., Rollet-Labelle, E., Naccache, P.H., 2002. Preservation of the pattern of tyrosine phosphorylation in human neutrophil

C. Gilbert et al. / Journal of Immunological Methods 271 (2002) 185–201 lysates: II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling. J. Immunol. Methods 261, 85 – 101. Hart, D., 1975. Evidence for the non-protein nature of the receptor for the enterotoxin in Vibrio cholerae on murine lymphoid cells. Infect. Immun. 11, 742 – 747. Ibarrola, I., Vossebeld, P.J., Homburg, C.H., Thelen, M., Roos, D., Verhoeven, A.J., 1997. Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa. Biochim. Biophys. Acta 1357, 348 – 358. Ignatoski, K.M., Verderame, M.F., 1996. Lysis buffer composition dramatically affects extraction of phosphotyrosine-containing proteins. BioTechniques 20, 794 – 796. Khamzina, L., Borgeat, P., 1998. Correlation of alpha-fetoprotein expression in normal hepatocytes during development with tyrosine phosphorylation and insulin receptor expression. Mol. Biol. Cell 9, 1093 – 1105. Marcilla, A., Rivero-Lezcano, O.M., Agarwal, A., Robbins, K.C., 1995. Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fc gamma receptors. J. Biol. Chem. 270, 9115 – 9120. McDonald, P.P., Bovolenta, C., Cassatella, M., 1998. Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon gamma and GM-CSF and necessity to overcome the action of endogenous proteases. Biochemistry 37, 13173 – 13265. Naccache, P.H., Gilbert, C., Barabe, F., Al-Shami, A., Mahana, W., Bourgoin, S.G., 1997. Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils. J. Leukoc. Biol. 62, 901 – 910. Papavassiliou, A.G., 1994. Preservation of protein phosphoryl groups in immunoprecipitation assays. J. Immunol. Methods 170, 67 – 73. Pouliot, M., McDonald, P.P., Krump, E., Mancini, J.A., McColl, S.R., Weech, P.K., Borgeat, P., 1996. Colocalization of cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils. Eur. J. Biochem. 238, 250 – 258.

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Pouliot, M., Gilbert, C., Borgeat, P., Poubelle, P.E., Bourgoin, S., Cre´minon, C., Maclouf, J., McColl, S.R., Naccache, P.H., 1998. Expression and activity of prostanglandin endoperoxide synthase-2 in agonist-activated human neutrophils. FASEB J. 12, 1109 – 1123. Rollet, E., Caon, A.C., Roberge, C.J., Liao, N.W., Malawista, S.E., McColl, S.R., Naccache, P.H., 1994. Tyrosine phosphorylation in activated human neutrophils. Comparison of the effects of different classes of agonists and identification of the signaling pathways involved. J. Immunol. 153, 353 – 363. Rollet-Labelle, E., Gilbert, C., Naccache, P.H., 2000. Modulation of human neutrophil responses to CD32 cross-linking by serine/ threonine phosphatase inhibitors: cross-talk between serine/ threonine and tyrosine phosphorylation. J. Immunol. 164, 1020 – 1028. Simons, K.T.D., 2000. Lipid rafts and signal transduction. Nat. Rev. 1, 31 – 39. Smith, J.A., 1994. Neutrophils, host defense, and inflammation: a double-edged sword. J. Leukoc. Biol. 56, 672. Solomon, K.R., Mallory, M.A., Finberg, R.W., 1998. Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells. Biochem. J. 334, 325 – 333. Welch, H., Maridonneau-Parini, I., 1997. Lyn and Fgr are activated in distinct membrane fractions of human granulocytic cells. Oncogene 15, 2021 – 2029. Yan, S.R., Fumagalli, L., Berton, G., 1996. Activation of SRC family kinases in human neutrophils. Evidence that p58CFGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity. FEBS Lett. 380, 198 – 203. Zhou, M.J., Lublin, D.M., Link, D.C., Brown, E.J., 1995. Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear leukocytes. Implications for immune complex activation of the respiratory burst. J. Biol. Chem. 270, 13553 – 13560.