IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry (0006-2960) 2004 Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu 08-DEC-2006: Contacted by JYOTI. yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu Accepted 2006-NOV-16 by DAVET. 08-DEC-2006: Contacted by JYOTI. Biochemistry (0006-2960) 2004 Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry (0006-2960) 2004 Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu 08-DEC-2006: Contacted by JYOTI. mouse-l cells Mus musculus embryo fibroblast, Moloney Sarcoma Virus transformed l cells Mouse embryo fibroblast, Moloney Sarcoma Virus transformed electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy immunostaining immunostaining Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu Accepted 2006-NOV-16 by DAVET. 08-DEC-2006: Contacted by JYOTI. Biochemistry (0006-2960) 2004 Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry (0006-2960) 2004 Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu 08-DEC-2006: Contacted by JYOTI. mouse-l cells Mus musculus embryo fibroblast, Moloney Sarcoma Virus transformed l cells Mouse embryo fibroblast, Moloney Sarcoma Virus transformed confocal microscopy confocal microscopy immunostaining immunostaining Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu Accepted 2006-NOV-16 by DAVET. 08-DEC-2006: Contacted by JYOTI. Biochemistry (0006-2960) 2004 Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry (0006-2960) 2004 Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu 08-DEC-2006: Contacted by JYOTI. mouse-l cells Mus musculus embryo fibroblast, Moloney Sarcoma Virus transformed l cells Mouse embryo fibroblast, Moloney Sarcoma Virus transformed anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer The experiments involving PITP could not be curated as the antibody recognised PITPalpha and beta. Also the authors do not specify which protein, alpha or beta, is used as the PITP construct. In addition, data for several other experiments is not shown. Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu Accepted 2006-NOV-16 by DAVET. 08-DEC-2006: Contacted by JYOTI. Biochemistry (0006-2960) 2004 Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry (0006-2960) 2004 Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu 08-DEC-2006: Contacted by JYOTI. mouse-l cells Mus musculus embryo fibroblast, Moloney Sarcoma Virus transformed l cells Mouse embryo fibroblast, Moloney Sarcoma Virus transformed fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret predetermined predetermined participant predetermined Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. The species of the SCP-2 protein was obtained by looking at a reference from one of the papers referenced (Seedorf et al. (1994) J Bio Chem 269 2613). The species of the caveolin-1 protein was inferred by looking at a publication by the donor of the construct. fschroeder@cvm.tamu.edu Accepted 2006-NOV-16 by DAVET. 08-DEC-2006: Contacted by JYOTI. Biochemistry (0006-2960) 2004 Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry (0006-2960) 2004 Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu 08-DEC-2006: Contacted by JYOTI. rat-mca_rh7777 Rattus norvegicus McA-RH7777 cells (hepatoma) mca_rh7777 buffalo rat hepatoma cells Species: rat, buffalo female; Tissue: liver; Tumor: hepatoma; Derived from: Morris hepatoma 7777 confocal microscopy confocal microscopy immunostaining immunostaining Zhou M., Parr RD., Petrescu AD., Payne HR., Atshaves BP., Kier AB., Ball JM., Schroeder F. Cancer - Interactions investigated in the context of cancer Mock-transfected cells were double immunolabeled with anti-sera to SCP-2 and caveolin- 1 followed by reaction with secondary antibodies conjugated to Rhodamine Red X and FITC, respectively. The cells were examined by laser scanning confocal microscopy. fschroeder@cvm.tamu.edu Accepted 2006-NOV-16 by DAVET. 08-DEC-2006: Contacted by JYOTI. Biochemistry (0006-2960) 2004 scp2_rat Sterol carrier protein 2 Propanoyl-CoA C-acyltransferase Sterol carrier protein X SCP-chi Scp2 Scp-2 Acetyl-CoA C-myristoyltransferase Non-specific lipid-transfer protein SCP-2/3-oxoacyl-CoA thiolase SCP-2/thiolase Straight-chain acyl-CoA oxidase protein protein rat Rattus norvegicus (Rat) Rat MPSVALNSPRLPRVFVVGVGMTKFMKPGGENSRDYPDLAKEAGQKALADRQIPYSAVEQACVGYVYGESTCGQRAIYHSLGLTGIPIINVNNNCSTGSTALFMAQQLVQGGLANCVLALGFEKMEKGSLGTKYSDRSNPLEKHIDVLINKYGMSACPFAPQLFGSAGKEHMETYGTKVEHFAKIGWKNHKHSVNNPYSQFQDEYSLDEIMKSRPVFDFLTVLQCCPTSDGAAAAIVSSEEFVQKHGLQSKAVEIVAQEMVTDMPSTFEEKSVIKMVGYDMSKEAARKCYEKSGLGPSDVDVIELHDCFSTNELLTYEALGLCPEGQGGALVDRGDNTYGGKWVINPSGGLISKGHPLGATGLAQCAELCWQLRGEAGKRQVPGAKVALQHNLGLGGAAVVTLYRMGFPEAASSFRTHQISAAPTSSAGDGFKANLIFKEIEKKLEEEGEEFVKKIGGIFAFKVKDGPGGKEATWVVDVKNGKGSVLPDSDKKADCTITMADSDLLALMTGKMNPQSAFFQGKLKIAGNMGLAMKLQSLQLQPDKAKL D0D1B435D2DC6AFB cav1_rat Caveolin-1 Cav1 Cav protein protein rat Rattus norvegicus (Rat) Rat MSGGKYVDSEGHLYTVPIREQGNIYKPNNKAMADEVNEKQVYDAHTKEIDLVNRDPKHLNDDVVKIDFEDVIAEPEGTHSFDGIWKASFTTFTVTKYWFYRLLSTIFGIPMALIWGIYFAILSFLHIWAVVPCIKSFLIEIQCISRVYSIYVHTFCDPLFEAIGKIFSNIRISTQEEI 55E8F6ABC7514FD2 cav1_mouse Caveolin-1 Cav1 Cav protein protein mouse Mus musculus Mouse MSGGKYVDSEGHLYTVPIREQGNIYKPNNKAMADEVTEKQVYDAHTKEIDLVNRDPKHLNDDVVKIDFEDVIAEPEGTHSFDGIWKASFTTFTVTKYWFYRLLSTIFGIPMALIWGIYFAILSFLHIWAVVPCIKSFLIEIQCISRVYSIYVHTFCDPLFEAIGKIFSNIRISTQKEI DIP protein P49817 original sequence version: 94 DIP protein P49817 original sequence version: 143 DIP protein P49817 original sequence version: 168 CF88FE9B1D3D67AE cav1_human Caveolin-1 CAV1 CAV protein protein human Homo sapiens Human MSGGKYVDSEGHLYTVPIREQGNIYKPNNKAMADELSEKQVYDAHTKEIDLVNRDPKHLNDDVVKIDFEDVIAEPEGTHSFDGIWKASFTTFTVTKYWFYRLLSALFGIPMALIWGIYFAILSFLHIWAVVPCIKSFLIEIQCISRVYSIYVHTVCDPLFEAVGKIFSNVRINLQKEI DIP protein Q03135 original sequence version: 164 DIP protein Q03135 original sequence version: 119 DIP protein Q03135 original sequence version: 122 DIP protein Q03135-1 original sequence version: 122 DIP protein Q03135 original sequence version: 133 DIP protein Q03135 original sequence version: 155 DIP protein Q03135 original sequence version: 147 2424DE9B5E6521D5 scp2_mouse Sterol carrier protein 2 Scp2 Scp-2 Propanoyl-CoA C-acyltransferase Sterol carrier protein X SCP-chi Acetyl-CoA C-myristoyltransferase Non-specific lipid-transfer protein SCP-2/3-oxoacyl-CoA thiolase SCP-2/thiolase Straight-chain acyl-CoA oxidase protein protein mouse Mus musculus Mouse MPSVALKSPRLRRVFVVGVGMTKFMKPGGENSRDYPDMAKEAGQKALEDAQIPYSAVEQACVGYVYGDSTSGQRAIYHSLGLTGIPIINVNNNCSTGSTALFMAHQLIQGGLANCVLALGFEKMERGSIGTKFSDRTTPTDKHIEVLIDKYGLSAHPITPQMFGYAGKEHMEKYGTKVEHFAKIGWKNHKHSVNNTYSQFQDEYSLEEVMKSKPVFDFLTILQCCPTSDGAAAAILSSEEFVQQYGLQSKAVEIVAQEMMTDLPSTFEEKSIIKVVGYDMSKEAARRCYEKSGLTPNDVDVIELHDCFSVNELITYEALGLCPEGQGGTLVDRGDNTYGGKWVINPSGGLISKGHPLGATGLAQCAELCWQLRGEAGKRQVPGAKVALQHNLGLGGAVVVTLYRMGFPEAASSFRTHQVSAAPTSSAGDGFKANLVFKEIEKKLEEEGEQFVKKIGGIFAFKVKDGPGGKEATWVVDVKNGKGSVLPNSDKKADCTITMADSDLLALMTGKMNPQSAFFQGKLKIAGNMGLAMKLQNLQLQPGKAKL 37BA2E730D9CB105 scp2_human Sterol carrier protein 2 Propanoyl-CoA C-acyltransferase Sterol carrier protein X SCP-chi SCPX SCP2 Acetyl-CoA C-myristoyltransferase Non-specific lipid-transfer protein SCP-2/3-oxoacyl-CoA thiolase SCP-2/thiolase Straight-chain acyl-CoA oxidase protein protein human Homo sapiens Human MSSSPWEPATLRRVFVVGVGMTKFVKPGAENSRDYPDLAEEAGKKALADAQIPYSAVDQACVGYVFGDSTCGQRAIYHSLGMTGIPIINVNNNCATGSTALFMARQLIQGGVAECVLALGFEKMSKGSLGIKFSDRTIPTDKHVDLLINKYGLSAHPVAPQMFGYAGKEHMEKYGTKIEHFAKIGWKNHKHSVNNPYSQFQDEYSLDEVMASKEVFDFLTILQCCPTSDGAAAAILASEAFVQKYGLQSKAVEILAQEMMTDLPSSFEEKSIIKMVGFDMSKEAARKCYEKSGLTPNDIDVIELHDCFSTNELLTYEALGLCPEGQGATLVDRGDNTYGGKWVINPSGGLISKGHPLGATGLAQCAELCWQLRGEAGKRQVPGAKVALQHNLGIGGAVVVTLYKMGFPEAASSFRTHQIEAVPTSSASDGFKANLVFKEIEKKLEEEGEQFVKKIGGIFAFKVKDGPGGKEATWVVDVKNGKGSVLPNSDKKADCTITMADSDFLALMTGKMNPQSAFFQGKLKITGNMGLAMKLQNLQLQPGNAKL 29F7551465C7143A cav1-scp2 3 11 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component colocalization colocalization 1 H, Table 1 in L-cells the colocalized SCP-2/caveolin-1 was significantly more concentrated at the plasma membrane In L-cell plasma membranes, 86.7 plus or minus 9.9% of SCP-2-pixels were colocalized with 87.6 plus or minus 10.9% of caveolin-1-pixels. Plasma membranes of cells containing little SCP-2 but much caveolin-1 (i.e., L-cells) exhibited 3.5-fold more colocalized SCP-2/caveolin-1 than plasma membranes of cells containing high levels of SCP-2 but relatively little caveolin-1 (i.e., hepatoma cells). However, in both cases at least part of SCP-2 was structurally colocalized with caveolin- 1, primarily at the plasma membrane in L-cells, and more so in punctate (vesicles) structures containing caveolin-1 in hepatoma cells. cav1-scp2-1 6 7 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component colocalization colocalization In hepatoma cells, the colocalized SCP-2/caveolin-1 (yellow pixels) were distributed primarily in distinct punctuate structures (Figure 1D) but less so at the plasma membrane 1 D, Table 1 Quantitative pixel fluorogram analysis within select plasma membrane regions showed that an average of 24.8 plus or minus 13.4% of SCP-2 -pixels were colocalized with 75.6 plus or minus 15.3% of caveolin-1-pixels within the hepatoma cell plasma membrane. Plasma membranes of cells containing little SCP-2 but much caveolin-1 (i.e., L-cells) exhibited 3.5-fold more colocalized SCP-2/caveolin-1 than plasma membranes of cells containing high levels of SCP-2 but relatively little caveolin-1 (i.e., hepatoma cells). However, in both cases at least part of SCP-2 was structurally colocalized with caveolin- 1, primarily at the plasma membrane in L-cells, and more so in punctate (vesicles) structures containing caveolin-1 in hepatoma cells. scp2-cav1 5 12 unspecified role unspecified role fluorescence donor fluorescence donor region Cy3 donor tag cy3 label cy3 label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region Cy5 acceptor tag cy5 label cy5 label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction 2 caveolin-1 detected by sensitized emission appeared distributed mostly at the plasma membranes of L-cells, clearly indicating that at the plasma membrane the SCP-2 molecules were very close to caveolin- 1 (i.e., within less than 100 �, the limit of FRET sensitivity) scp2-cav1-1 2 12 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component colocalization colocalization Human SCP-2 was overexpressed in L-cells. The intermolecular distance between 15 nm gold particles (detecting SCP-2) and 6 nm gold particles (detecting caveolin-1) determined in six examples from several cells ranged from about 100-240 �. 3 A, B The data indicate SCP-2 and caveolin-1 colocalize within the plasma membrane in regions well within the size of caveolae. The distance between the proteins as determined by immunogold electron microscopy is greater than for FRET as the colloidal gold particles are larger than Cy3/Cy5, there is repulsion between the gold particles and fewer measurements were taken. scp2-cav1-2 4 9 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait physical association physical association The L-cells overexpressed human SCP-2. 8 Anti-SCP-2 antibody successfully coimmunoprecipitated caveolin-1 cav1-scp2-2 4 12 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association The L-cells overexpressed human SCP-2. 8 Anti-caveolin-1 antibody successfully precipitated caveolin-1 (Figure 8B, lane 3) and coprecipitated SCP-2 scp2-cav1-3 1 10 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait physical association physical association 12, Table 2 the yeast two-hybrid assay demonstrated a protein-protein interaction between SCP-2 and caveolin-1 in vivo cav1-scp2-3 1 12 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association 12, Table 2 the yeast two-hybrid assay demonstrated a protein-protein interaction between SCP-2 and caveolin-1 in vivo