Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5

Protein Expression and PuriWcation 53 (2007) 1–8 www.elsevier.com/locate/yprep

Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5 Jie Yang a, Shi-Ying Guo a, Fei-Yan Pan a, Hui-Xia Geng a, Yi Gong a, Dan Lou a, Yong-Qian Shu b,¤, Chao-Jun Li a,c,¤ a

Jiangsu Key Laboratory for Molecular & Medical Biotechnology, Life Science College, Nanjing Normal University, Nanjing 210097, China b Oncology Department, AYliated No. 1 Hospital of Nanjing Medical University, Nanjing 210029, China c Animal Model Research Center, Nanjing University, Nanjing 210095, China Received 8 August 2006, and in revised form 23 September 2006 Available online 14 November 2006

Abstract Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We Wrst identiWed mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, puriWcation, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a speciWc antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. ImmunoXuorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5. © 2006 Elsevier Inc. All rights reserved. Keywords: mRabL5; Rab-like; Prokaryotic expression; Polyclonal antibody

Introduction Rab proteins represent a large subfamily of the Ras superfamily including many kinds of GTP-binding proteins which play key roles in the secretory and endocytic pathways in eukaryotic cells ranging from yeast to mammals. The molecular weight of Rab ranges from 20 to 25 kDa [1]. Although some Rabs have a more restricted tissue distribution, many are ubiquitously expressed [2]. Now it is believed that Rabs are engaged in multiple aspects of membrane biology, including regulation of vesicle budding, docking, motility along cytosketal systems, and membrane fusion by

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Corresponding authors. Fax: +86 25 8372 4440 (Y.-Q. Shu), +86 25 8359 8812 (C.-J. Li). E-mail addresses: [email protected] (Y.-Q. Shu), licj@ njnu.edu.cn (C.-J. Li). 1046-5928/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.10.025

interconverting between a GTP-binding form and a GDPbinding form [3,4] under the inXuence of additional factors such as GTPase activating proteins (GAPs),1 GDP dissociation inhibitors (GDIs), and the guanosine exchange factors (GEFs) [5–7]. Human cells contain nearly 70 Rabs and Rab-like proteins [8,9] and each is believed to be speciWcally associated with a distinct membrane-bound compartment or pathway. For example, Rab5 is localized on early endosomes; Rab6 is on the Golgi complex; and Rab7 and Rab9 are on late endosomes. The speciWcity of Rab localization is determined by the

1 Abbreviations used: GAPs, GTPase activating proteins; GDIs, GDP dissociation inhibitors; GEFs, guanosine exchange factors; ORF, open reading frame; IPTG, isopropyl--D-thiogalactopyranoside; RT, room temperature; IPP, Image Pro-plus; ELISA, enzyme-linked immunosorbent assay; OPD, o-phenylendiamine.

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structural characteristic unique to each family member [10– 13]. Such structural determinants appear to be recognized by distinct sets of proteins on organelle surfaces [13–17]. Since protein transport is such a complicated multistep process, it can be predicted that more Rab proteins exist to regulate the vesicular traYc. The investigation of novel Rab proteins will bring better outcomes to the study of membrane traYc. Here mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, was proved to be present as a Golgi-associated protein (unpublished data). In the present work, we prepared two polyclonal antibodies against the full length of mRabL5 and the last 14 amino acids residues of mRabL5 C terminus, respectively. These antibodies can be used to elucidate the function of mRabL5 on vesicular traYc and its molecular mechanism. Materials and methods Cloning of mRabL5 and constructions of expression vector Full-length cDNA of mRabL5 (GenBank Accession No. NM_026073) is composed of 931 bp and contained a complete open reading frame (ORF) of 558 bp potentially encoding a 185-residue Rab-like protein. The mRabL5 coding sequence was ampliWed by PCR from the T/A-mRabL5 plasmid constructed by our lab. Oligos correspond to the N-terminal (5⬘-TCC CCC GGG TCT GAA GGC TAA GAT C-3⬘) and C-terminal (5⬘-CCG CTC GAG GGT GAT GAT AAG CAT CTC-3⬘) regions, containing the SmaI and XhoI restriction sites, respectively (underlined). The reaction was carried out using the following reaction cycles in a Peltier Thermal Cycler (MJ Research, USA): initial denaturation at 95 °C for 3 min followed by 35 consecutive cycles of denaturation at 95 °C for 40 s, annealing for 1 min at 55 °C, extension at 72 °C for 2 min, and then Wnal extension at 72 °C for 7 min followed. The ampliWed mRabL5 gene was gel-puriWed by high pure PCR product puriWcation kit (Axygen Biotechnology, Hangzhou, China). After digestion with SmaI and XhoI, the puriWed product was inserted into corresponding region of pGEX-4T-1 expression vector (Amersham Biosciences, Piscataway, NJ) and conWrmed by restriction enzyme digestion and sequencing. The correct recombinant prokaryotic expression vector was named as pGEX-4T-mRabL5. In order to raise a more speciWc polyclonal serum in rabbits, we synthesized (Invitrogen, Shanghai, China) the coding sequence based on the last 14 amino acids of mRabL5 C terminus (M S E S R D R E E M L I I T, amino acid residues from 172 to 185) with 5⬘ EcoRI and 3⬘ XhoI digestion sites, and then subcloned it into pGEX-4T-1 expression vector. The correct recombinant was conWrmed by sequencing and named pGEX-4T-mRabL5-C. Cultivation and induction conditions Escherichia coli strain Rosetta cells were transformed with recombined plasmids, pGEX-4T-mRabL5 and

pGEX-4T-1-mRabL5-C, respectively. Rosetta, the derivational strain of BL21, contains the extra gene copies for coding rare tRNA and facilitates the prokaryotic expression of eukaryotic protein in E. coli. The GST fusion protein was induced with diVerent concentrations of IPTG (0.05, 0.1, 1 mM) under various conditions of induction time (2, 3, 4 h), temperature (20, 30, 37 °C) and cell density (OD600 D 0.6, 0.8, 1.0). After optimizing the best induction condition for obtaining soluble fusion protein as much as possible, the GST fusion genes were expressed on a large scale as follows. The transformants were cultured in 25 ml LB medium containing 100 g/ml ampicillin and grown overnight at 37 °C and 250 rpm. These pre-inocula were then transferred to 500 ml LB medium containing ampicillin of the same concentration. The cultures were grown at 37 °C and 250 rpm until OD600 was 1.0. Expression of the GST fusion protein was induced with 0.1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 2.5 h at 30 °C. Extraction of GST fusion proteins The GST fusion proteins were extracted as described previously [18] with some modiWcations. BrieXy, the cells were harvested at 4 °C by centrifugation at 6000g for 10 min. The pellets were suspended (3 ml/g wet weight) in lysis buVer (50 mM Tris–HCl buVer, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1.0 mM PMSF and 1.0 mg/ml lysozyme). The suspensions were incubated for 20 min at 4 °C with stirring, and then 0.04 mg of deoxycholic acid (Sigma–Aldrich, St. Louis, Missouri, USA) was added. The cell suspension was sonicated on ice. The resulting cell lysate was centrifuged at 12,000 rpm for 15 min. The clear supernatant (soluble fraction) was collected and the remaining pellet (insoluble fraction) which contains inclusion bodies was also resuspended in equal volume of lysis buVer. Soluble and insoluble fractions were then analyzed on 12% SDS–PAGE. PuriWcation of GST fusion proteins GST fusion proteins were puriWed by aYnity chromatography (Glutathione Sepharose™ 4 Fast Flow, Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s protocol for batch puriWcation with a little improvement. BrieXy, wash 100 l Glutathione Sepharose 4 Fast Flow with PBS twice. Incubate the cell lysate with the prepared Glutathione Sepharose 4 Fast Flow for 20–30 min at room temperature (RT) with end-over-end rotation. Sediment the resin by centrifugation at 500g for 5 min and the supernatant was carefully decanted. The resin was washed with PBS for three times. The bound protein was eluted by adding 100 l elution buVer (50 mM Tris–HCl, 10 mM reduced glutathione, pH 8.0) and incubating at RT for 5–10 min. Sediment the beads by centrifugation at 500g for 5 min. Carefully collect the supernatant (eluted protein). The concentration of GST fusion protein can be estimated by measuring the absorbance at 280 nm: A280 » 1 corre-

J. Yang et al. / Protein Expression and PuriWcation 53 (2007) 1–8

sponds to »0.5 mg/ml. Eluted material from the gel was monitored for GST fusion proteins using SDS–PAGE to determine the puriWcation eYciency. Gel electrophoresis To conWrm and compare the quality of recombinant protein expression, SDS–PAGE electrophoresis of the proteins was performed as described by Sambrook et al. [18], using 12% acrylamide gels followed by stained with Coomassie brilliant blue overnight and destained in 6% acetic acid until a clear background was reached, and then analyzed with Image Pro-plus (IPP) software. Production of polyclonal antibodies against the recombinant protein The puriWed recombinant proteins were used for raising antibodies in New Zealand white rabbits according to standard protocol [19]. Rabbit was Wrst immunized subcutaneously using 500 g of recombinant protein in Freund’s complete adjuvant. After two weeks, the rabbit was boosted intramuscularly twice with 250 g recombinant protein each in incomplete Freund’s adjuvant at one-week interval. Before every immunization, blood samples were taken from the marginal vein of the rabbit ear, centrifuged, and the sera were obtained to determine the antibody titer by ELISA. PuriWcation of rabbit antisera The anti-mRabL5 and anti-mRabL5 C terminus sera were puriWed using protein G aYnity chromatography (Roche Diagnositcs GmbH, Mannheim, Germany) according to the protocol for cross-linking IgG to protein G (NEB, Ipswich, MA). Wash 100 l beads with PBS for three times. Add 80 l PBS (0.1 M) and 15–25 l serum to the beads. Mix thoroughly and incubate at 4 °C with agitation for 30 min. The supernatant was carefully decanted after centrifugation at 500g for 5 min. The beads were washed for three times with PBS, and then incubated at RT for 5–10 min in 30 l elution buVer containing 0.1 M glycine– HCl (pH 2.5) with end-over-end rotation. Harvest beads by centrifugation at 500g for 5 min and collect the supernatant (puriWed antibody). Antiserum titer determination by ELISA The titers of antiserum were examined using an indirect enzyme-linked immunosorbent assay (ELISA). In short, the wells of polystyrene microtiter plates (Greiner Bio-One, USA) were coated with 150 l antigen (50 g/ml). After incubation overnight at 4 °C, the wells were washed three times with PBS–Tween buVer (0.05% Tween 20 in PBS). The coated wells were blocked with 200 l of 3% BSA for 1 h at 37 °C and then incubated with 150 l polyclonal antibodies against mRabL5 or mRabL5 C terminus with diVerent deliquations (from 1:500 to 1:25600). After incubation

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for 2 h at 37 °C, the wells were incubated with 150 l horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution 1:10000, Sigma, USA) for 1 h at 37 °C after thoroughly washed. OPD (o-phenylendiamine, Shanghai Chemicals, China) was used as the substrate of peroxidase. After 15 min, the reaction was stopped by 50 l H2SO4 (2 M) and the absorbance was measured at 490 nm using an ELx800 Microplate Reader (Bio-Tek Instruments, Inc. Winooski, Vermont, USA). Cell culture and recombinant adenovirus infection 293A cells and NIH3T3 cells were grown in Dulbecco’s modiWed Eagle’s medium (Gibco, Grand Island, New York) supplemented with 10% NCS (HyClone, Hampton, NH), 2 mM L-glutamine, 20 U/ml penicillin, and 20 mg/ml streptomycin. The cultures were kept in a 5% CO2 and 95% air humidiWed incubator at 37 °C. Adenoviruses expressing mRabL5 (prepared by our lab) were incubated with cells in a small volume of serum free medium at 37 °C. After adsorption for 2 h, fresh complete growth medium was added and the cells were placed in the incubator for the following experiments. IdentiWcation of anti-mRabL5 and anti-mRabL5 C terminus antisera by Western blot 293A cells overexpressing mRabL5 by adenovirus infection were harvested, and then lysed in cell lysis buVer (20 mM Tris–HCl, pH 8.0, 75 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1.0% NP-40) containing proteolyses enzyme inhibitors. Western blot analysis was performed according to the standard procedure. After 12% SDS–PAGE electrophoresis, the gel was immersed in the transfer buVer (0.24% Tris–HCl, 1.153% glycine, and 15% methanol, pH 8.8), and the proteins were transferred to PVDF membrane by electrophoresis at 80 V for 1.5 h. The membrane was incubated for 1 h in blocking buVer (3% BSA in PBS) at RT. After being washed three times (10 min each) with PBS–Tween buVer, the membrane was incubated with the anti-mRabL5 or anti-mRabL5 C terminus (1:1500 diluted) polyclonal antibody overnight at 4 °C. The membrane was then incubated for 1 h with alkaline phosphatase (Ap)-conjugated goat anti-rabbit IgG (BOSTER, Wuhan, China, 1:1000 diluted) at RT after thoroughly washed. The speciWc protein bands were visualized with 5-bromo-4-chloro-3- indolylphosphate and nitroblue tetrazolium. ImmunoXuorescence For immunoXuorescence staining, NIH3T3 cells, without any exogenous gene expression, grown on sterile coverslips were washed with PBS, and then Wxed with cold 3.7% paraformaldehyde in PBS for 30 min at RT. After Wxation, cells were washed with PBS for 5 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min at RT, and further incubated in blocking buVer (3% BSA in PBS) for 1 h at RT. Cells were

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Fig. 1. Construction of pGEX-4T-mRabL5. (A) Schematic diagram of the ORF cloned in pGEX-4T-1 expression vector. (B) IdentiWcation of recombinant plasmid pGEX-4T-mRabL5 by restriction digestion. Lane 1, pGEX-4T-mRabL5/SmaI and XhoI; lane 2, pGEX-4T-mRabL5; lane 3, pGEX-4T-1; M, DNA marker of 2000 bp.

then incubated with antisera (1:200 dilution) at 4 °C overnight. After rinsing cells three times with PBS–Tween buVer (5 min each), they were incubated with the goat anti-Rabbit Rhodamine-conjugated secondary antibody in PBS at 30 °C for 2 h. Cells were then washed three times with PBS–Tween buVer (5 min each). After staining nuclear DNA with DAPI for 15 min at RT, cells were distinguished under a Xuorescence microscope and photographed with a Spot Cool CCD (Diagnostic Instruments MI). Results Cloning of mRabL5 and mRabL5-C in pGEX-4T-1 expression vector The complete cDNA of mRabL5 (GenBank Accession No. NM_026073) was ampliWed by nest PCR from the mouse 9.5d mice embryo. On the basis of complete cDNA of mRabL5, we designed primers for ampliWcation of this gene from T/AmRabL5 plasmid (prepared by our lab). SmaI and XhoI sites were designed in the primers to facilitate cloning in pGEX-4T1 expression vector (Fig. 1A). The PCR product was ligated to pGEX-4T-1 vector. The clones were screened by PCR and restriction digestion (Fig. 1B). The cloned gene was conWrmed by sequencing and named pGEX-4T-mRabL5. Meanwhile, the coding sequence based on the last 14 amino acid residues of mRabL5 C terminus with 5⬘ EcoRI and 3⬘ XhoI sites was synthesized and subcloned into pGEX4T-1 expression vector. After conWrming by sequencing, the correct plasmid was designated as pGEX-4T-mRabL5-C. Expression of the recombinant proteins Cloned mRabL5 gene and the coding sequence of mRabL5 C terminus in pGEX-4T-1, under the control of

IPTG-inducible tac promotor, are predicted to encode recombinant proteins with molecular weights of »46 and »27 kDa, respectively. The conWrmed constructs were transformed into Rosetta host cells. Small-scale cultures were Wrst subjected to IPTG induction to identify the capacity of expression. By SDS–PAGE analysis, correct recombinant proteins with predicted molecular weights of »46 and »27 kDa were selectively expressed in the transformed E. coli Rosetta cells (Fig. 2A, lanes 3 and 6 arrowed) and apparently constituted a large fraction (more than 50%, calculated by IPP software) of the total protein. Overexpressed protein was not found without induction of E. coli Rosetta cells transformed with the pGEX-4T-mRabL5 or pGEX-4T-mRabL5C constructs (Fig. 2A, lanes 2 and 5) nor was it detected in the negative control E. coli Rosetta cells (Fig. 2A, lane 8). In order to obtain soluble fusion proteins as much as possible, we tried to optimize the expression conditions as described in material and methods. We found that fusion protein solubility can be dramatically increased by lowering the growth temperature during induction combining with the induction at a higher cell density for a short period of time. Finally, an induction condition of 0.1 mM IPTG, 2 h, 30 °C with a cell density at OD600 D 1.0 was chosen for a large scale cultivation. By SDS–PAGE analysis and IPP software calculation, approximately 80% of GST-mRabL5 and more than 95% of GST-mRabL5 C terminus were detected in soluble fraction, respectively (Fig. 2B). PuriWcation of the recombinant protein The soluble fusion proteins bound to the resin were eluted with elution buVer (Fig. 3, lanes 4 and 8). The protein yields were 6 and 4.6 mg/L of cell culture for full length mRabL5 fusion protein and C terminus mRabL5 fusion protein, respectively.

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Fig. 2. Expression of GST fusion proteins. (A) Expression of GST-mRabL5 (lanes 1–3) and GST-mRabL5 C terminus (lanes 4–6) by SDS–PAGE analysis. Proteins were separated on 12% acrylamide gel and stained with Coomassie blue. Lanes 1 and 4, cells carrying the vector with the insert before IPTG induction; lanes 2 and 5, total cellular protein without IPTG treatment during induction; lanes 3 and 6, total cellular protein after IPTG induction for 2 h; lanes 7 and 8, cells without vector before (lane 7) or after (lane 8) IPTG induction. (B) Optimization of expression conditions to obtain soluble proteins.

antibody (Fig. 5, lane 2). By the same Western blot analysis, we found that the anti-mRabL5 C terminus antiserum shows qualitatively equivalent recognition of mRabL5 compared with anti-mRabL5 antiserum (Fig. 5, lanes 3 and 4), which indicated that both antibodies can serve as a good tool to characterize mRabL5 protein. Detection of endogenous mRabL5 by immunoXuorescence staining

Fig. 3. PuriWcation of GST-mRabL5 (lanes 1–4) and GST-mRabL5 C terminus (lanes 5–8). Lanes 1 and 5, cells carrying the vector with the insert before IPTG induction; lanes 2 and 6, total cell proteins after IPTG induction; lanes 3 and 7, soluble fractions of total cell lysate; lanes 4 and 8, puriWed recombinant proteins.

Titer and speciWcity analysis by ELISA and Western blot After immunizing rabbits with full length mRabL5 fusion protein or C terminus mRabL5 fusion protein respectively according to standard protocol, anti-mRabL5 serum and anti-mRabL5 C terminus serum were puriWed by protein G aYnity chromatography as described in material and methods. Both antibody titers were determined by ELISA and were found to be approximately 1:32000 (Fig. 4). At the same time, preimmunized rabbit serum used as a negative control did not result in a detectable signal. Western blot analysis with the polyclonal antibodies raised against the full length mRabL5 fusion protein detected a »20 kDa protein in mRabL5 overexpressing 293A cell line lysate (Fig. 5, lane 1). The GST-mRabL5 fusion protein could also be recognized by the polyclonal

ImmunoXuorescence staining result suggested that the polyclonal antibody we prepared could eVectively recognize endogenous mRabL5 and display its subcellular distribution as a perinuclear clustering pattern in NIH3T3 cell line (Fig. 6). Anti-mRabL5 C terminus serum had less nonspeciWc signal than that of full length mRabL5 (Fig. 6D). Discussion With the development of Genome Projects, more and more novel Rab proteins are being characterized in detail. Some of them are being named Rab-like proteins which display features that diverge from the typical Rab proteins. Such diVerences may represent more recent evolutionary modiWcations of the Rab family [20,21]. At the same time, emerging evidence implicates alterations in the Rab GTPases and their associated regulators and eVectors in multiple human diseases. So the investigation of novel Rab proteins as well as the physiologic and pathophysiologic roles of Rab proteins may cast new light on the study of the protein transport and bring better outcomes to therapeutic intervention [22]. In the present work, as an eVort to obtain a good tool to elucidate the functional involvement of mRabL5, a novel M. musculus Rab-like protein involved in the cellular mem-

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Fig. 4. Determination of antibody titers by ELISA. Null-1, without antisera. Null-2, without primal antigen and antisera.

brane traYc (our unpublished data), we cloned, expressed, puriWed mRabL5, and then raised the polyclonal antibodies against the novel Rab-like protein. Protein blast analysis revealed that the less conserved regions were present at the C terminus of mRabL5, while the full length of mRabL5 showed homology with some other Rab proteins. So in order to obtain a more speciWc antibody against mRabL5, we also prepared the polyclonal antibody against the last 14 amino acid residues of mRabL5 C terminus. Western blot

and immunoXuorescence results suggested that both antibodies can recognize mRabL5 and serve as a good tool for its biological investigation, although anti-mRabL5 C terminus antiserum had less nonspeciWc signal compared with that of full length mRabL5 by immunoXuorescence analysis (Fig. 6). In the initial stage, E. coli strains Rosetta and M15 were both chosen as host cells to perform prokaryotic expression. GST fusion proteins were not successfully

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Fig. 5. Western blot analysis of the speciWcity of antisera. Lanes 1 and 2, incubated with anti-mRabL5 antiserum as primary antibody; lanes 3 and 4, incubated with anti-mRabL5 C terminus antiserum as primary antibody; lanes 1 and 3, 293A cell lysate overexpressed with mRabL5 by adenovirus infection; lanes 2 and 4, GST-mRabL5 fusion protein.

expressed in M15 strain due to the rare codons in the coding sequence of mRabL5 which impede the expression of eukaryotic protein in M15 strain. But by supplying rare tRNAs, the Rosetta strain provide for “universal” translation and enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. Using Rosetta as host cell, we optimized the cul-

ture and induction parameters to get more soluble fusion proteins. We found that an induction condition of 0.1 mM IPTG for 2 h at 30 °C with a cell density at OD600 D 1.0 was the optimal system for the prokaryotic expression of mRabL5. Our Wnding suggested that both antibodies can recognize mRabL5 protein in eucaryotic cell lysate, while the

Fig. 6. ImmunoXuorescence analysis of mRabL5. Endogenous mRabL5 in NIH3T3 cell line was detected using rabbit anti-mRabL5 serum (A, arrowed, red) and anti-mRabL5 C terminus serum (D, arrowed, red) as primary antibody, respectively, and a goat anti-Rabbit Rhodamine-conjugated secondary antibody. Nuclear DNA was stained with DAPI (B, E, blue). The merged pictures showed in C and F. (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this paper.)

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anti-mRabL5 C terminus antiserum has less nonspeciWc signal for mRabL5 detection and could serve as a tool to identify mRabL5 subcellular localization, and may further open the doors to understand its functional involvements in endocytic and exocytic pathways. Acknowledgments We thank T.C. He and B. Vogelstein for the plasmid pAdTrack-CMV and pAdEasy-1, and W. Ning for giving information results of hypoxic SAGE library of HAECs, respectively. References [1] F. Schimmoller, I. Simon, S.R. PfeVer, Rab GTPases, Directors of vesicle docking, J. Biol. Chem. 273 (1998) 22161–22164. [2] S.R. PfeVer, Structural clues to Rab GTPase functional diversity, J. Biol. Chem. 280 (2005) 15485–15488. [3] C. Walch-Solimena, R.N. Collins, P.J. Novick, Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of postGolgi vesicles, J. Cell Biol. 137 (1997) 1495–1509. [4] H. Horiuchi, R. Lippe, H.M. McBride, M. Rubino, P. Woodman, H. Stenmark, V. Rybin, M. Wilm, K. Ashman, M. Mann, M. Zerial, A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to eVector recruitment and function, Cell 90 (1997) 1149–1159. [5] K. Simons, M. Zerial, Rab proteins and the road maps for intracellular transport, Neuron 11 (1993) 789–799. [6] L. Gonzalez Jr., R.H. Scheller, Regulation of membrane traYcking: structural insights from a Rab/eVector complex, Cell 96 (1999) 755–758. [7] J.S. Rodman, N. Wandinger-Ness, Rab GTPases coordinate endocytosis, J. Cell Sci. 113 (2000) 183–192. [8] J. Colicelli, Human RAS Superfamily Proteins and Related GTPases, Sci. STKE 2004 (2004), re13. [9] M. Zerial, H. McBride, Rab proteins as membrane organizers, Nat. Rev. Mol. Cell Biol. 2 (2001) 107–117.

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