Quantitative Proteomics of Caveolin-1-regulated Proteins
2010; Elsevier BV; Volume: 9; Issue: 10 Linguagem: Inglês
10.1074/mcp.m110.001289
ISSN1535-9484
AutoresAlberto Dávalos, Carlos Fernández‐Hernando, Grzegorz Sowa, Behrad Derakhshan, Michelle I. Lin, Ji Y. Lee, Hongyu Zhao, Ruiyan Luo, Christopher M. Colangelo, William C. Sessa,
Tópico(s)Caveolin-1 and cellular processes
ResumoCaveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs. Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs. Caveolae have been extensively studied and shown to play an important role in the regulation of many cellular functions including cell signaling, vesicular transport, and lipid metabolism. Caveolae are especially abundant in adipocytes, endothelial cells (ECs), 1The abbreviations used are:ECendothelial cellCavcaveolinPTRFpolymerase I and transcript release factorDRMdetergent-resistant membraneSDPRserum deprivation response proteinNSnegative nonsilencing controlHUVEChuman umbilical vein ECiTRAQisobaric tagging for relative and absolute quantificationMBSMes-buffered salineIPIInternational Protein IndexeNOSendothelial nitric-oxide synthaseRCreconstitutionDSPdithiobis(succinimidylpropionate)IPimmunoprecipitation. 1The abbreviations used are:ECendothelial cellCavcaveolinPTRFpolymerase I and transcript release factorDRMdetergent-resistant membraneSDPRserum deprivation response proteinNSnegative nonsilencing controlHUVEChuman umbilical vein ECiTRAQisobaric tagging for relative and absolute quantificationMBSMes-buffered salineIPIInternational Protein IndexeNOSendothelial nitric-oxide synthaseRCreconstitutionDSPdithiobis(succinimidylpropionate)IPimmunoprecipitation. fibroblasts, and smooth muscle cells (1Parton R.G. 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There are three isoforms of caveolin, caveolin-1 (Cav-1), Cav-2, and Cav-3, that have been extensively studied (1Parton R.G. Simons K. The multiple faces of caveolae.Nat. Rev. Mol. Cell Biol. 2007; 8: 185-194Crossref PubMed Scopus (1128) Google Scholar, 4Cohen A.W. Hnasko R. Schubert W. Lisanti M.P. Role of caveolae and caveolins in health and disease.Physiol. Rev. 2004; 84: 1341-1379Crossref PubMed Scopus (714) Google Scholar). Cav-1 and Cav-3 are required for caveola formation in different tissues (8Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1291) Google Scholar, 9Fra A.M. Williamson E. Simons K. Parton R.G. De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.Proc. Natl. Acad. Sci. 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Although the roles of caveolins in caveola formation are well known, the requirement of other proteins regulating caveolin function and thus caveola formation has been recently addressed (12Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 13Liu L. Brown D. McKee M. Lebrasseur N.K. Yang D. Albrecht K.H. Ravid K. Pilch P.F. Deletion of Cavin/PTRF causes global loss of caveolae, dyslipidemia, and glucose intolerance.Cell Metab. 2008; 8: 310-317Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 14Liu L. Pilch P.F. A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization.J. Biol. 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To identify proteins that stably interact and are regulated by Cav-1, we used an unbiased proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ). iTRAQ is a powerful quantitative proteomics tool for the relative quantification of proteins in complex mixtures by mass spectrometry (15Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3615) Google Scholar), and this technology has been successfully used for global comparison of protein expression in many cells systems and in different tissues (16Guo Y. Singleton P.A. Rowshan A. Gucek M. Cole R.N. Graham D.R. Van Eyk J.E. Garcia J.G. Quantitative proteomics analysis of human endothelial cell membrane rafts: evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induced barrier enhancement.Mol. Cell. Proteomics. 2007; 6: 689-696Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Ralhan R. Desouza L.V. Matta A. Chandra Tripathi S. Ghanny S. Datta Gupta S. Bahadur S. Siu K.W. Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry.Mol. Cell. Proteomics. 2008; 7: 1162-1173Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). As caveolae and Cav-1 are present in the endothelium lining all blood vessels of the body (18Frank P.G. Woodman S.E. Park D.S. Lisanti M.P. Caveolin, caveolae, and endothelial cell function.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1161-1168Crossref PubMed Scopus (291) Google Scholar, 19Stan R.V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (176) Google Scholar) and play an important role in the regulation of several EC functions (8Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1291) Google Scholar, 20Murata T. Lin M.I. Huang Y. Yu J. Bauer P.M. Giordano F.J. Sessa W.C. Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice.J. Exp. Med. 2007; 204: 2373-2382Crossref PubMed Scopus (183) Google Scholar, 21Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 22Yu J. Bergaya S. Murata T. Alp I.F. Bauer M.P. Lin M.I. Drab M. Kurzchalia T.V. Stan R.V. Sessa W.C. Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels.J. Clin. Investig. 2006; 116: 1284-1291Crossref PubMed Scopus (273) Google Scholar), we examined the protein expression profile of proteins localized in detergent-resistant membranes (DRMs) prepared from the lungs of wild-type (WT) and Cav-1 KO mice via iTRAQ labeling and multidimensional LC and MS/MS analysis. Here, we show that Cav-1 was required for the appearance of Cav-2, polymerase I and transcript release factor (PTRF)/Cavin-1, and serum deprivation response protein (SDPR; also known as Cavin-2) in isolated DRMs from lungs, confirming the initial discovery of these proteins as regulators of Cav-1-dependent caveola function. Isolated as a functional complex in cells, Cavin-1 co-fractionated into DRMs with Cav-1 and -2 but Cavin-1 formed high molecular weight homo-oligomers separated from Cav-1 and Cav-2 using sedimentation velocity gradients. Knockdown of Cavin-1 in ECs affected several functions including nitric oxide production, migration, and morphogenesis. Thus, Cavin-1 has a critical role in regulating several aspects of caveola function in ECs. Membrane rafts were isolated as described previously (23Abrami L. Fivaz M. Kobayashi T. Kinoshita T. Parton R.G. van der Goot F.G. Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.J. Biol. Chem. 2001; 276: 30729-30736Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) with minor modifications. Briefly, lungs from three mice (female, 8–10 weeks old) were isolated, and tissue was minced with scissors on ice and transferred into a tube. Lungs were adjusted up to a 3-ml volume with cold Mes-buffered saline (MBS; 25 mm Mes, pH 6.5, 150 mm NaCl) containing protease inhibitors and homogenized using a Dounce homogenizer. The homogenate was adjusted to 3.25 ml with MBS, and 3.25 ml of MBS containing 2% Triton X-100 was added and homogenized. The sample was rocked for 30 min at 4 °C. Next, the suspension was mixed with an equal volume of 85% sucrose in MBS. 2 ml of the 42.5% sucrose suspension was overlaid with 8 ml of 35% sucrose and 2 ml of 5% sucrose in an ultracentrifuge tube and centrifuged for 18 h at 35,000 rpm at 4 °C in an SW40 rotor. 12 fractions of 1 ml were collected from the top and mixed with SDS-PAGE loading buffer, and equal volumes were loaded, separated by SDS-12% PAGE, and Western blotted. For iTRAQ analysis, fractions 1 and 2 were pooled together, sucrose was diluted to ½ with cold water, and the pooled sample was split in SW28 rotor ultracentrifuge tubes and centrifuged for 90 min at 28,000 rpm at 4 °C. The pellets were pooled, precipitated with chloroform:methanol, and submitted to iTRAQ analysis. 200 µg of protein pellets was dissolved in the solution buffer and reduced, and cysteines were blocked as described in the protocol of the iTRAQTM kit (Applied Biosystems). In total, three biological replicates were performed, and each 4-plex iTRAQ had an experimental (technical) replicate for both the wild-type and knock-out samples. Briefly, after overnight trypsin digestion, 100 µg of the samples was labeled with iTRAQ tags as follow: two WT samples with iTRAQ 114 and iTRAQ 115 reagents, respectively, and two Cav-1 KO samples with iTRAQ 116 and iTRAQ 117 reagents, respectively. Labeled samples were pooled and purified using a strong cation exchange column (Applied Biosystems) and separated into 20 fractions. For QSTAR XL LC-MS/MS analysis, each cation exchange fraction was dried and resuspended in 10 µl of 0.1% formic acid in preparation for reverse phase LC with the LC Packings UltiMate work station, allowing us to preconcentrate the 10-µl samples on a Waters 5-mm C18 Symmetry 300 trap column. The individual peptides were then separated at a flow rate of 450 nl/min on an in-line 100-µm × 15-cm Waters Atlantis C18 column equilibrated with 0.5% acetic acid, 5% acetonitrile and eluted with a 60-min acetonitrile gradient. Data collection was performed by electrospray ionization of the eluent with data-dependent acquisition on an Applied Biosystems API QSTAR XL mass spectrometer. Each of the QSTAR XL mass spectrometer spectra files (*.wiff) was processed with Mascot Distiller version 2.1, the resulting peak lists were combined, and the database was searched using Mascot Server 2.1. The search parameters included trypsin with one miss cleavage, static modifications carbamidomethyl (Cys) and iTRAQ reagents (N terminus and Lys), and variable modification oxidation (Met). Data analysis on the resulting LC-MS and MS/MS data sets was accomplished using a dual processor Dell 650 Workstation. The search results for each fraction were analyzed using the IPI mouse 3.27 database (released March 27, 2007 and contained 53,831 sequences). After Mascot analysis, PeptideProphet and ProteinProphet (Institute for Systems Biology) analyses (24Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3796) Google Scholar, 25Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3527) Google Scholar) were performed using the Trans-Proteomic Pipeline version 2.7 MIST Revision 2, Build 200601091318. PeptideProphet and ProteinProphet compute the probabilities for both individually searched peptides and the resulting proteins, respectively. The protein validation was performed using both PeptideProphet and ProteinProphet values such that a protein was validated if it had at least two top ranked peptides with each peptide probability score above 95% and above 94%, respectively (supplemental Fig. S1). The ProteinProphet probability cutoff corresponded to a 0.004, 0.003, and 0.003 false positive error rate for all three biological replicates (supplemental Table S1). Finally, all Trans-Proteomic Pipeline identifications were submitted to Yale Proteomics Expression Database web site for further user analysis. iTRAQ quantitation and secondary protein identification were performed using the ParagonTM search algorithm (26Shilov I.V. Seymour S.L. Patel A.A. Loboda A. Tang W.H. Keating S.P. Hunter C.L. Nuwaysir L.M. Schaeffer D.A. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.Mol. Cell. Proteomics. 2007; 6: 1638-1655Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar) in the ProteinPilot 2.0 software. The IPI mouse 3.27 database (released March 27, 2007) was used using "thorough search." The "iTRAQ 4-plex peptide-labeled" sample type and a "biological modification ID focus" were selected in the analysis method. Trypsin was selected as the digestion enzyme with cysteine alkylation by methyl methanethiosulfonate as a modification. Raw data that included, but were not limited to, reporter ion peak areas, reporter ion peak area error, peptide assignment, and confidence were exported from ProteinPilot (tab-delimited) without the ProteinPilot autobias correction applied (non-normalized data) so we could perform quantile normalization as described below. The tab-delimited ProteinPilot results were then uploaded into our Yale Proteomics Expression Database. For secondary protein identification, each protein had to have also been identified by Paragon and had to have two or more identified peptides and a ProteinPilot confidence score >2 (99% confidence level). To ensure that we only compared data that ProteinPilot includes in its quantitation analysis, peptides had to be classified as "used," which requires the presence of an iTRAQ label and at least one valid iTRAQ reporter ion ratio. Additionally, ProteinPilot only uses high quality reporter ions for the peak area measurements to calculate iTRAQ peptide ratios. To remove low intensity reporter ion ratios from the exported raw data sets, we used the non-modifiable criterion of ProteinPilot that requires that valid iTRAQ reporter ion ratios must contain two iTRAQ reporter ions with a summed signal to noise ratio >9 to be included in the analysis. All raw data (mzXML), peak list (mgf), Mascot search results (dat), ProteinPilot search results, PeptideProphet (pepXML), and ProteinProphet (protXML) files are publicly available through http://yped.med.yale.edu/repository (access code, aSKezE). The Yale Proteomics Expression Database has additional features, which enabled us to perform sample comparisons and Panther classification. Quantile normalization of the iTRAQ data was performed to correct the variation of the protein abundance using the statistical package R. Principal component analysis was used to examine the consistency of the technical replicates and biological replicates. Among 425 unique proteins observed in the experiments, there were 117 proteins identified in common through the experiments. A cutoff was set at 0.8 for down-regulated proteins and at 1.2 for up-regulated proteins as reported for other works (27Salim K. Kehoe L. Minkoff M.S. Bilsland J.G. Munoz-Sanjuan I. Guest P.C. Identification of differentiating neural progenitor cell markers using shotgun isobaric tagging mass spectrometry.Stem Cells Dev. 2006; 15: 461-470Crossref PubMed Scopus (25) Google Scholar, 28Seshi B. An integrated approach to mapping the proteome of the human bone marrow stromal cell.Proteomics. 2006; 6: 5169-5182Crossref PubMed Scopus (47) Google Scholar). Protein expression values from technical and biological replicates were analyzed under the analysis of variance model. Nitric oxide production was compared using t test and pairwise comparisons by the Mann-Whitney test. Effects on protein or gene expression were analyzed by analysis of variance, and comparisons between each experimental condition and the control were made by the confidence interval method. All values are presented as the mean ± S.E., and a p value of less than 0.05 was considered statistically significant. Calculations were performed using GraphPad Prism 4 software (San Diego, CA) or SigmaStat Statistical Analysis, System Version 1.00 (Jandel Corp., San Rafael, CA). Mouse SDPR antibody was a kind gift from Prof. R. G. W. Anderson (University of Texas Southwestern Medical Center). The following antibodies were obtained from commercial sources: rabbit anti-caveolin (610060, BD Biosciences), mouse anti-caveolin 2 (610685, BD Biosciences), mouse anti-HSP90 (610419, BD Biosciences), mouse anti-endothelial nitric-oxide synthase (eNOS) (610297, BD Biosciences), mouse anti-PTRF (611259, BD Biosciences), rabbit anti-PTRF (A301-271A and A301-270A, Bethyl Laboratories), rabbit anti-phospho-eNOS (36-9100; Zymed Laboratories Inc.), rabbit anti-NOS (sc-653, Santa Cruz Biotechnology), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-caveolin-1 (NB 100-615, Novus Biologicals), and mouse anti-SDPR (H08436-B01, Novus Biologicals). COS-7, HEK293, and EAhy.926 were maintained in high glucose DMEM supplemented with 10% FBS; l-glutamine; antibiotics; and hypoxanthine, aminopterin, and thymidine supplement (EAhy.926) at 37 °C in a humidified atmosphere of 5% CO2. Human umbilical ECs (HUVECs) were maintained in M199 medium, and only passages 2–3 were used for experiments. Mouse lung endothelial cells isolated from WT, Cav-1 KO, Cav-1 RC, and Cav-1 transgenic mice were maintained in EBM-2 medium supplemented with EGM-2 MV SingleQuots. After dissection, aortas (from 8-week-old mice) were fixed with 4% paraformaldehyde for 10 min at 4 °C and then dehydrated in 15% sucrose overnight at 4 °C. The vessels were then embedded in OCT (Sakura) and frozen. Serial 10-µm sections were blocked with 3% goat serum. Slides were incubated with either rabbit anti-Cavin-1 (Bethyl Laboratories) or rabbit anti-Cav-1 antibody (BD Biosciences) at a 1:100 dilution each at 4 °C overnight. Alexa Fluor 488 or 594 anti-rabbit IgG (Invitrogen) was used as the secondary antibody (1:250 dilution at room temperature for 1 h). For cells, COS-7 and EA.hy.926 cells grown on coverslips were fixed with 4% paraformaldehyde for 5 min, rinsed with PBS, permeabilized with 0.1% Triton X-100 for 10 min, washed with PBS, and blocked with 5% goat serum for 45 min at room temperature. Cells were incubated with the primary antibodies (diluted 1:200) overnight at 4 °C and washed twice with blocking solution followed by a 45-min incubation with appropriate secondary antibodies conjugated to immunofluorescent dye Alexa Fluor 488 or Alexa Fluor 594 (diluted 1:250) at room temperature. After washing three times, coverslips were mounted on slides with gelvatol/DAPI (Sigma-Aldrich) and analyzed with an epifluorescence microscope (Axiovert, Carl Zeiss MicroImaging, Inc.). Images were acquired using a charge-coupled device camera (Axio, Carl Zeiss MicroImaging, Inc.). Analysis of different images was performed using OpenLab software (Improvision) after subtracting background. Total RNA was extracted with TRIzol reagent using RNeasy columns (Qiagen). Reverse transcription was performed using 2 µg of total RNA using TaqMan reverse transcription reagents (Applied Biosystem), and quantitative PCR was performed using iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer's protocol on an iCycler quantitative PCR analyzer (Bio-Rad). The Cavin-1 target sequence against 5′-CAACTTTAAAGTCATGATCTA-3′ was obtained from Qiagen (high performance-guaranteed siRNA), and a scrambled siRNA was used as a negative nonsilencing control (NS) (5′-AATTCTCCGAACGTGTCACGT-3′). ECs at 50% confluence were transfected with RNAi construct (75 nm) using Oligofectamine (Invitrogen) according to the manufacturer's instructions for 8 h in Opti-MEM (Invitrogen) and then incubated in full medium for 48 h. A second 8 h of transfection was performed, and then either HUVEC or EAhy.926 growth medium was added for an additional 48 h. After reaching 40% confluence in a 6-well tissue culture plate, HEK293 or COS-7 cells were transfected with 1 µg of cDNA plasmid for 8 h with FuGENE 6 reagent in Opti-MEM. Then, medium was replaced with DMEM, 10% FBS for 36 h. cDNAs for human HA-tagged Cav-1 or Myc-tagged Cav-2 were constructed and subcloned in pcDNA3 vector (Invitrogen). Cells (150-mm dish) were washed twice with PBS; scraped into 2 ml of ice-cold 500 mm sodium carbonate, pH 11 supplemented with 1 mg/ml protease inhibitor mixture (Roche Applied Science); homogenized using a Dounce homogenizer; and sonicated (three 20-s bursts at 30% maximal power). The homogenate was then adjusted to 42.5% sucrose by the addition of 2 ml of 85% sucrose prepared in 25 mm Mes, pH 6.5, 0.5 m NaCl and placed at the bottom of an ultracentrifuge tube. A 5–30% discontinuous sucrose gradient was formed (3 ml of 5% sucrose and 5 ml of 30% sucrose, both in Mes containing 250 mm sodium carbonate) and centrifuged at 35,000 rpm for 18 h in an SW40 rotor (Beckman Coulter). 12 gradient fractions (1 ml) were collected from the top and mixed with SDS-PAGE loading buffer, and equal volumes were loaded, separated by SDS-12% PAGE, and Western blotted. The percentage of total proteins in different fractions was determined by densitometry and plotted as percentage of total protein (NIH Image program). Lysed cells were solubilized with either 60 mm octyl glucoside or 1% Triton X-100 for 1 h at 4 °C, and immunoprecipitation was performed on 500 µg of total cell protein using antibodies or nonspecific normal rabbit IgG (4 µg) for 2 h at 4 °C. Protein G-Sepharose (Sigma) was then added and incubated for 1 h. Beads were precipitated by centrifugation, and the supernatant was collected. The immune complexes were washed three times with immunoprecipitation buffer, and both the supernatants and immunoprecipitates were boiled in SDS sample buffer before resolving by SDS-PAGE and Western blotting using the antibodies described above. The influence of Cavin in caveolin oligomerization was analyzed as described for caveolin-1 (29Sargiacomo M. Scherer P.E. Tang Z. Kübler E. Song K.S. Sanders M.C. Lisanti M.P. Oligomeric structure of caveolin: implications for caveolae membrane organization.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9407-9411Crossref PubMed Scopus (472) Google Scholar) and caveolin-2 (30Sowa G. Pypaert M. Fulton D. Sessa W.C. The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6511-6516Crossref PubMed Scopus (84) Google Scholar) with minor modifications. Briefly, sample were dissociated with 700 µl of MBS plus 60 mm n-octyl-β-d-glucopyranoside). Solubilized material was then loaded atop a 5–50% linear sucrose gradient (12 ml) and centrifuged at 42,000 rpm for 16 h in an SW40 rotor (Beckman Coulter). After centrifugation, 12 1-ml gradient fractions were collected from the top and mixed with SDS-PAGE loading buffer, and equal volumes were loaded, separated by SDS-12% PAGE, and W
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