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/ http://www.ebi.ac.uk/ ivan-2001-2 HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting western blot western blot Immuno blot Immuno blot Ivan M., Kondo K., Yang H., Kim W., Valiando J., Ohh M., Salic A., Asara J.M., Lane W.S., Kaelin W.G. Cloning, expression and purification of the recombinant VHL-elonginB-elonginC complex used as probe (detected by anti-VHL antibody) in the far western experiments is described in PMID:10205047. william_kaelin@dfci.harvard.edu Science (0036-8075) 2001 Only protein-protein interactions imex curation ivan-2001-1 HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. in vitro In vitro pull down pull down autoradiography autoradiography Ivan M., Kondo K., Yang H., Kim W., Valiando J., Ohh M., Salic A., Asara J.M., Lane W.S., Kaelin W.G. william_kaelin@dfci.harvard.edu Science (0036-8075) 2001 Only protein-protein interactions imex curation ivan-2001-4 HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. mouse-ts20 Mus musculus mouse-ts20 Thermosensitive E1 ubiquitin-activating enzyme embryonic fibroblast cell mutant A31N-ts20 anti bait coip anti bait coimmunoprecipitation anti bait coip autoradiography autoradiography Ivan M., Kondo K., Yang H., Kim W., Valiando J., Ohh M., Salic A., Asara J.M., Lane W.S., Kaelin W.G. ts20 cells were stably transfected to produce HA-pVHL were immunoprecipitated with anti–HIF-1a (lane 1 and 2) or anti-HA (lanes 3 to 7). Bound proteins were eluted by boiling in sample buffer (lane 1 and 2) or treatment with the indicated peptides, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. william_kaelin@dfci.harvard.edu Science (0036-8075) 2001 Only protein-protein interactions imex curation ivan-2001-3 HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. in vitro In vitro pull down pull down autoradiography autoradiography Ivan M., Kondo K., Yang H., Kim W., Valiando J., Ohh M., Salic A., Asara J.M., Lane W.S., Kaelin W.G. Cloning, expression and purification of the GST-tagged (on VHL) recombinant VHL-elonginB-elonginC complex used as bait is described in PMID:10205047. The construct containing Gal4 DNA-binding domain, HA tag and Hif1-alpha is described in PMID:10878807. william_kaelin@dfci.harvard.edu Science (0036-8075) 2001 Only protein-protein interactions imex curation vhl_human von Hippel-Lindau disease tumor suppressor VHL pVHL Protein G7 protein protein human Homo sapiens Human MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD epas1_human Endothelial PAS domain-containing protein 1 EPAS1 HIF2A MOP2 Member of PAS protein 2 Basic-helix-loop-helix-PAS protein MOP2 BHLHE73 PASD2 Hypoxia-inducible factor 2-alpha HIF-1-alpha-like factor PAS domain-containing protein 2 Class E basic helix-loop-helix protein 73 protein protein human Homo sapiens Human MTADKEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLAISFLRTHKLLSSVCSENESEAEADQQMDNLYLKALEGFIAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENLSLKNGSGFGKKSKDMSTERDFFMRMKCTVTNRGRTVNLKSATWKVLHCTGQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQHPSHMDIPLDSKTFLSRHSMDMKFTYCDDRITELIGYHPEELLGRSAYEFYHALDSENMTKSHQNLCTKGQVVSGQYRMLAKHGGYVWLETQGTVIYNPRNLQPQCIMCVNYVLSEIEKNDVVFSMDQTESLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTKLKEEPEELAQLAPTPGDAIISLDFGNQNFEESSAYGKAILPPSQPWATELRSHSTQSEAGSLPAFTVPQAAAPGSTTPSATSSSSSCSTPNSPEDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAAPLGPPVSPPHVSTFKTRSAKGFGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQDLSGGDPPGGSTSHLMWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHLPLPQPPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGPSFESYLLPELTRYDCEVNVPVLGSSTLLQGGDLLRALDQAT hif1a_mouse Hypoxia-inducible factor 1-alpha Hif1a ARNT-interacting protein protein protein mouse Mus musculus Mouse MEGAGGENEKKKMSSERRKEKSRDAARSRRSKESEVFYELAHQLPLPHNVSSHLDKASVMRLTISYLRVRKLLDAGGLDSEDEMKAQMDCFYLKALDGFVMVLTDDGDMVYISDNVNKYMGLTQFELTGHSVFDFTHPCDHEEMREMLTHRNGPVRKGKELNTQRSFFLRMKCTLTSRGRTMNIKSATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMTCLVLICEPIPHPSNIEIPLDSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYNTKNSQPQCIVCVNYVVSGIIQHDLIFSLQQTESVLKPVESSDMKMTQLFTKVESEDTSCLFDKLKKEPDALTLLAPAAGDTIISLDFGSDDTETEDQQLEDVPLYNDVMFPSSNEKLNINLAMSPLPSSETPKPLRSSADPALNQEVALKLESSPESLGLSFTMPQIQDQPASPSDGSTRQSSPERLLQENVNTPNFSQPNSPSEYCFDVDSDMVNVFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESNSPSPPSMSTVTGFQQTQLQKPTITATATTTATTDESKTETKDNKEDIKILIASPSSTQVPQETTTAKASAYSGTHSRTASPDRAGKRVIEQTDKAHPRSLNLSATLNQRNTVPEEELNPKTIASQNAQRKRKMEHDGSLFQAAGIGTLLQQPGDCAPTMSLSWKRVKGFISSEQNGTEQKTIILIPSDLACRLLGQSMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN hif1a_human Hypoxia-inducible factor 1-alpha ARNT-interacting protein Member of PAS protein 1 Basic-helix-loop-helix-PAS protein MOP1 HIF1A MOP1 BHLHE78 PASD8 PAS domain-containing protein 8 Class E basic helix-loop-helix protein 78 protein protein human Homo sapiens Human MEGAGGANDKKKISSERRKEKSRDAARSRRSKESEVFYELAHQLPLPHNVSSHLDKASVMRLTISYLRVRKLLDAGDLDIEDDMKAQMNCFYLKALDGFVMVLTDDGDMIYISDNVNKYMGLTQFELTGHSVFDFTHPCDHEEMREMLTHRNGLVKKGKEQNTQRSFFLRMKCTLTSRGRTMNIKSATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMTCLVLICEPIPHPSNIEIPLDSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYNTKNSQPQCIVCVNYVVSGIIQHDLIFSLQQTECVLKPVESSDMKMTQLFTKVESEDTSSLFDKLKKEPDALTLLAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQQTQIQEPTANATTTTATTDELKTVTKDRMEDIKILIASPSPTHIHKETTSATSSPYRDTQSRTASPNRAGKGVIEQTEKSHPRSPNVLSVALSQRTTVPEEELNPKILALQNAQRKRKMEHDGSLFQAVGIGTLLQQPDDHAATTSLSWKRVKGCKSSEQNGMEQKTIILIPSDLACRLLGQSMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN vhl-epas1-1 1453973 n/a 1453977 unspecified role unspecified role bait bait vhl_tumour_suppress_b/a_dom sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) n/a 1453978 unspecified role unspecified role prey prey human-786_o Homo sapiens renal cell adenocarcinoma 786_o Human renal cell adenocarcinoma cell line CRL-1932 is the ATCC identifier. ATCC identifier is CRL-1932. association association false false false Fig. 1a Increasing amounts of cobalt chloride (CoCl2) (2, 10, 100, 1000 micromolar) or desferrioxamine (DFO) (2, 10, 100, 1000 micromolar) inhibits the interaction. vhl-hif1a-6 1453973 n/a 1453979 unspecified role unspecified role prey prey mouse-ts20 Mus musculus mouse-ts20 Thermosensitive E1 ubiquitin-activating enzyme embryonic fibroblast cell mutant A31N-ts20 n/a 1453977 unspecified role unspecified role bait bait vhl_tumour_suppress_b/a_dom sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) association association false false false Fig. 1b Hypoxic condition (1% oxygen) inhibits the interaction. vhl-hif1a-7 1453973 n/a 1453977 unspecified role unspecified role bait bait vhl_tumour_suppress_b/a_dom sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) n/a 1453980 unspecified role unspecified role prey prey region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobd Escherichia coli (strain B / BL21-DE3) association association false false false Fig. 1c, Fig. 1d The interaction only occurs after bacterially expressed Hif1-alpha is incubated with rabbit reticulocyte lysate (for 90 minutes at 30C). Heat-inactivated rabbit reticulocyte lysate does not have this effect. hif1a-vhl-5 1453974 n/a 1453977 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false in vitro In vitro n/a 1453980 unspecified role unspecified role bait bait deg_odph_vhl_1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false required hydroxypro-564 4hypro 4-hydroxy-L-proline (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid 4-hydroxy-L-proline 4-hydroxylated L-proline 4-hydroxyproline 4-trans-hydroxy-L-proline L-threo-4-hydroxyproline MOD_RES 4-hydroxyproline gamma-hydroxypyrrolidine-alpha-carboxylic acid 4HyPro modified residue ms mass detection of residue modification modified residue ms certain certain sequence position certain certain certain sequence position certain false hydroxylation of pro564 region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q16665:p.Pro564Ala mutation disrupting mutation disrupting interaction mutation disrupting alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false Q16665:p.Leu562Ala mutation disrupting mutation disrupting interaction mutation disrupting alanine scanning alanine scanning certain certain sequence position certain certain certain sequence position certain false Q16665:p.[Met561_Leu562delinsAlaAla;Pro564_Met568delinsAlaAlaAlaAlaAla] mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false Fig. 2b, Fig. 4, Fig 5a, Fig. 5b The interaction only occurs after chemically synthesized Hif1-alpha peptides are incubated with rabbit reticulocyte lysate. cul2-hif1a 1453974 n/a 1453980 unspecified role unspecified role bait bait deg_odph_vhl_1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q16665:p.Pro564Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false required hydroxypro-564 4hypro 4-hydroxy-L-proline (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid 4-hydroxy-L-proline 4-hydroxylated L-proline 4-hydroxyproline 4-trans-hydroxy-L-proline L-threo-4-hydroxyproline MOD_RES 4-hydroxyproline gamma-hydroxypyrrolidine-alpha-carboxylic acid 4HyPro certain certain sequence position certain certain certain sequence position certain false hydroxylation of pro564 chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1453977 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-786_o Homo sapiens renal cell adenocarcinoma 786_o Human renal cell adenocarcinoma cell line CRL-1932 is the ATCC identifier. ATCC identifier is CRL-1932. physical association physical association false false false Fig. 5d The hydroxylated HIF(556-575) peptide specifically bound to pVHL as well as to proteins with the expected electrophoretic mobilities of the pVHL-associated proteins elongin B, elongin C, and Cul2. vhl-hif1a-9 1453975 n/a 1453980 competitor competitor competitor prey prey deg_odph_vhl_1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q16665:p.Pro564Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false required hydroxypro-564 4hypro 4-hydroxy-L-proline (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid 4-hydroxy-L-proline 4-hydroxylated L-proline 4-hydroxyproline 4-trans-hydroxy-L-proline L-threo-4-hydroxyproline MOD_RES 4-hydroxyproline gamma-hydroxypyrrolidine-alpha-carboxylic acid 4HyPro certain certain sequence position certain certain certain sequence position certain false hydroxylation of pro564 n/a 1453979 unspecified role unspecified role prey prey n/a 1453977 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Fig. 5c vhl-hif1a-10 1453975 n/a 1453977 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 1453979 unspecified role unspecified role prey prey physical association physical association false false false Fig. 5ci vhl-hif1a-8 1453976 n/a 1453977 unspecified role unspecified role bait bait vhl_tumour_suppress_b/a_dom sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecobd Escherichia coli (strain B / BL21-DE3) n/a 1453980 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false region gal4 dna bd gal4 dna binding domain gal4 dna bd undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false Q16665:p.Leu562Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false Q16665:p.Pro564Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false in vitro In vitro physical association physical association false false false Fig. 2a, Fig. 2c, Fig. 2d