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/ Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions human-hct116 Human colon carcinoma HCT116 cells hct116 Human colon carcinoma line HCT116 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis I put etoposide as an antagonist as it causes the complex to dissociate - yes sorry you are right xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference fluorescence fluorescence technology fluorescence immunostaining immunostaining Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis U2OS cells were treated with ALLN for 4 h or left untreated. (ALLN stabilises Mdm2). Cells were stained with a rabbit anti-Daxx and a mouse anti-Mdm2 antibodies. xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions in vitro In vitro pull down pull down ms participant Identification by mass spectrometry ms participant Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis HeLa S3 lysates were incubated with M2 beads (control) or M2 beads coated with recombinant Flag�Daxx. The bound proteins were resolved on SDS�PAGE and visualized by Coomassie blue staining. The 135 K protein band in the Flag�Daxx lane was identified as Hausp by mass spectrometry xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions mouse-embryonic_stem cell Mus musculus embryonic stem cell es cells embryonic stem cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis Wild-type and Daxx-deficient ES cells were treated with 5 uM MG-132 for 6 h. Cell lysates were immunoprecipitated with an anti- Hausp antibody and the control antibody. xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions mouse-embryonic_fibroblast Mus musculus embryonic fibroblasts embryonic_fibroblast Embryonic fibroblast Fibroblasts taken from an embryonic as opposed to a foetal or adult source. Foetus is defined as the stage at which tissue diferentiation is initiated. Tissue taken from an embryonic as opposed to a foetal or adult source. Embryo is defined as the development stage prior to organ formation. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis Lysate from p53-negative, Mdm2-negative MEFs were used for coimmunoprecipitation. xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. confocal microscopy confocal microscopy immunostaining immunostaining Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis Have used confocal microscopy and immunostaining as agreed xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions mouse-embryonic_fibroblast Mus musculus embryonic fibroblasts embryonic_fibroblast Embryonic fibroblast Fibroblasts taken from an embryonic as opposed to a foetal or adult source. Foetus is defined as the stage at which tissue diferentiation is initiated. Tissue taken from an embryonic as opposed to a foetal or adult source. Embryo is defined as the development stage prior to organ formation. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis It should be S4 E. Lysate from p53-negative, Mdm2-negative MEFs were used for coimmunoprecipitation. xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions human-h1299 Homo sapiens lung lymph node carcinoma h1299 Human lung lymph node carcinoma p53 null. These cells have also been reported to secrete the peptide hormone neuromedin B (NMB), but not gastrin releasing peptide (GRP). Also known as NCI H1299 or CRL-5803. http://www.lgcpromochem.com/atcc/ ATCC number: CRL-5803. ATCC number of the cell line is CRL-5803. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis HA�Hausp and GST�Mdm2 were cotransfected into H1299 in the presence or absence of Flag�Daxx. Cells were treated with 20 uM MG-132 for 4 h and the lysates were immunoprecipitated with M2 beads (lanes 3 and 4). Flag�Daxx and its binding complex were eluted using Flag peptide and the eluant was subject to a secondary immunoprecipitation with either anti-Mdm2 or a control antibody. Lysate from p53-negative, Mdm2-negative MEFs were used for coimmunoprecipitation. xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation Critical role for Daxx in regulating Mdm2. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. (1465-7392) 2006 imex curation Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis BioCreative - Critical Assessment of Information Extraction systems in Biology xyang@mail.med.upenn.edu 24-JAN-2007: Contacted by JYOTI. dave Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Tang J., Qu LK., Zhang J., Wang W., Michaelson JS., Degenhardt YY., El-Deiry WS., Yang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xyang@mail.med.upenn.edu Accepted 2006-SEP-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2006 dave Only protein-protein interactions imex curation p53_human Cellular tumor antigen p53 TP53 P53 Tumor suppressor p53 Phosphoprotein p53 Antigen NY-CO-13 protein protein human Homo sapiens Human MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD DIP protein P04637 original sequence version: 121 DIP protein P04637 original sequence version: 115 DIP protein P04637-1 original sequence version: 224 DIP protein P04637 original sequence version: 117 DIP protein P04637-1 original sequence version: 226 DIP protein P04637 original sequence version: 226 DIP protein P04637 original sequence version: 212 DIP protein P04637 original sequence version: 220 DIP protein P04637-1 original sequence version: 220 DIP protein P04637-1 original sequence version: 145 DIP protein P04637 original sequence version: 146 DIP protein P04637 original sequence version: 145 DIP protein P04637 original sequence version: 166 DIP protein P04637-1 original sequence version: 166 DIP protein P04637 original sequence version: 169 DIP protein P04637 original sequence version: 177 DIP protein P04637 original sequence version: 175 DIP protein P04637-1 original sequence version: 175 DIP protein P04637-1 original sequence version: 172 DIP protein P04637-1 original sequence version: 169 DIP protein P04637 original sequence version: 170 DIP protein P04637 original sequence version: 172 DIP protein P04637-1 original sequence version: 194 DIP protein P04637 original sequence version: 196 DIP protein P04637 original sequence version: 185 DIP protein P04637-1 original sequence version: 191 DIP protein P04637 original sequence version: 191 DIP protein P04637 original sequence version: 180 DIP protein P04637-1 original sequence version: 197 DIP protein P04637 original sequence version: 183 DIP protein P04637-1 original sequence version: 183 DIP protein P04637-1 original sequence version: 185 DIP protein P04637 original sequence version: 199 DIP protein P04637 original sequence version: 201 DIP protein P04637 original sequence version: 189 DIP protein P04637 original sequence version: 235 DIP protein P04637 original sequence version: 230 DIP protein P04637 original sequence version: 234 DIP protein P04637-1 original sequence version: 234 DIP protein P04637-1 original sequence version: 235 DIP protein P04637 original sequence version: 239 DIP protein P04637-1 original sequence version: 212 DIP protein P04637-1 original sequence version: 213 DIP protein P04637 original sequence version: 213 DIP protein P04637 original sequence version: 206 DIP protein P04637-1 original sequence version: 188 DIP protein P04637-1 original sequence version: 228 DIP protein P04637 original sequence version: 208 DIP protein P04637 original sequence version: 240 DIP protein P04637-1 original sequence version: 240 DIP protein P04637 original sequence version: 204 DIP protein P04637-1 original sequence version: 204 DIP protein P04637-1 original sequence version: 209 DIP protein P04637-1 original sequence version: 203 DIP protein P04637 original sequence version: 221 DIP protein P04637-1 original sequence version: 152 DIP protein P04637 original sequence version: 217 DIP protein P04637-1 original sequence version: 140 DIP protein P04637 original sequence version: 215 DIP protein P04637 original sequence version: 214 DIP protein P04637-1 original sequence version: 182 DIP protein P04637 original sequence version: 182 DIP protein P04637 original sequence version: 188 DIP protein P04637 original sequence version: 192 DIP protein P04637-1 original sequence version: 192 DIP protein P04637 original sequence version: 218 DIP protein P04637-1 original sequence version: 218 AD5C149FD8106131 ubp7_human Ubiquitin carboxyl-terminal hydrolase 7 USP7 HAUSP Ubiquitin thioesterase 7 Ubiquitin-specific-processing protease 7 Deubiquitinating enzyme 7 Herpesvirus-associated ubiquitin-specific protease protein protein human Homo sapiens Human MNHQQQQQQQKAGEQQLSEPEDMEMEAGDTDDPPRITQNPVINGNVALSDGHNTAEEDMEDDTSWRSEATFQFTVERFSRLSESVLSPPCFVRNLPWKIMVMPRFYPDRPHQKSVGFFLQCNAESDSTSWSCHAQAVLKIINYRDDEKSFSRRISHLFFHKENDWGFSNFMAWSEVTDPEKGFIDDDKVTFEVFVQADAPHGVAWDSKKHTGYVGLKNQGATCYMNSLLQTLFFTNQLRKAVYMMPTEGDDSSKSVPLALQRVFYELQHSDKPVGTKKLTKSFGWETLDSFMQHDVQELCRVLLDNVENKMKGTCVEGTIPKLFRGKMVSYIQCKEVDYRSDRREDYYDIQLSIKGKKNIFESFVDYVAVEQLDGDNKYDAGEHGLQEAEKGVKFLTLPPVLHLQLMRFMYDPQTDQNIKINDRFEFPEQLPLDEFLQKTDPKDPANYILHAVLVHSGDNHGGHYVVYLNPKGDGKWCKFDDDVVSRCTKEEAIEHNYGGHDDDLSVRHCTNAYMLVYIRESKLSEVLQAVTDHDIPQQLVERLQEEKRIEAQKRKERQEAHLYMQVQIVAEDQFCGHQGNDMYDEEKVKYTVFKVLKNSSLAEFVQSLSQTMGFPQDQIRLWPMQARSNGTKRPAMLDNEADGNKTMIELSDNENPWTIFLETVDPELAASGATLPKFDKDHDVMLFLKMYDPKTRSLNYCGHIYTPISCKIRDLLPVMCDRAGFIQDTSLILYEEVKPNLTERIQDYDVSLDKALDELMDGDIIVFQKDDPENDNSELPTAKEYFRDLYHRVDVIFCDKTIPNDPGFVVTLSNRMNYFQVAKTVAQRLNTDPMLLQFFKSQGYRDGPGNPLRHNYEGTLRDLLQFFKPRQPKKLYYQQLKMKITDFENRRSFKCIWLNSQFREEEITLYPDKHGCVRDLLEECKKAVELGEKASGKLRLLEIVSYKIIGVHQEDELLECLSPATSRTFRIEEIPLDQVDIDKENEMLVTVAHFHKEVFGTFGIPFLLRIHQGEHFREVMKRIQSLLDIQEKEFEKFKFAIVMMGRHQYINEDEYEVNLKDFEPQPGNMSHPRPWLGLDHFNKAPKRSRYTYLEKAIKIHN DIP protein Q93009 original sequence version: 71 DIP protein Q93009 original sequence version: 67 DIP protein Q93009-1 original sequence version: 180 DIP protein Q93009 original sequence version: 173 DIP protein Q93009 original sequence version: 91 DIP protein Q93009 original sequence version: 163 DIP protein Q93009 original sequence version: 176 DIP protein Q93009-1 original sequence version: 128 DIP protein Q93009 original sequence version: 128 F1A5A5C421396E45 daxx_human Death domain-associated protein 6 DAXX BING2 DAP6 Daxx Fas death domain-associated protein ETS1-associated protein 1 protein protein human Homo sapiens Human MATANSIIVLDDDDEDEAAAQPGPSHPLPNAASPGAEAPSSSEPHGARGSSSSGGKKCYKLENEKLFEEFLELCKMQTADHPEVVPFLYNRQQRAHSLFLASAEFCNILSRVLSRARSRPAKLYVYINELCTVLKAHSAKKKLNLAPAATTSNEPSGNNPPTHLSLDPTNAENTASQSPRTRGSRRQIQRLEQLLALYVAEIRRLQEKELDLSELDDPDSAYLQEARLKRKLIRLFGRLCELKDCSSLTGRVIEQRIPYRGTRYPEVNRRIERLINKPGPDTFPDYGDVLRAVEKAAARHSLGLPRQQLQLMAQDAFRDVGIRLQERRHLDLIYNFGCHLTDDYRPGVDPALSDPVLARRLRENRSLAMSRLDEVISKYAMLQDKSEEGERKKRRARLQGTSSHSADTPEASLDSGEGPSGMASQGCPSASRAETDDEDDEESDEEEEEEEEEEEEEATDSEEEEDLEQMQEGQEDDEEEDEEEEAAAGKDGDKSPMSSLQISNEKNLEPGKQISRSSGEQQNKGRIVSPSLLSEEPLAPSSIDAESNGEQPEELTLEEESPVSQLFELEIEALPLDTPSSVETDISSSRKQSEEPFTTVLENGAGMVSSTSFNGGVSPHNWGDSGPPCKKSRKEKKQTGSGPLGNSYVERQRSVHEKNGKKICTLPSPPSPLASLAPVADSSTRVDSPSHGLVTSSLCIPSPARLSQTPHSQPPRPGTCKTSVATQCDPEEIIVLSDSD 1B309ADDAA878040 mdm2_human E3 ubiquitin-protein ligase Mdm2 MDM2 p53-binding protein Mdm2 Oncoprotein Mdm2 Double minute 2 protein RING-type E3 ubiquitin transferase Mdm2 protein protein human Homo sapiens Human MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP DIP protein Q00987 original sequence version: 93 DIP protein Q00987-1 original sequence version: 192 DIP protein Q00987 original sequence version: 88 DIP protein Q00987 original sequence version: 180 DIP protein Q00987 original sequence version: 188 DIP protein Q00987 original sequence version: 189 DIP protein Q00987 original sequence version: 115 DIP protein Q00987-1 original sequence version: null DIP protein Q00987 original sequence version: 149 DIP protein Q00987 original sequence version: 146 DIP protein Q00987 original sequence version: 140 DIP protein Q00987 original sequence version: 183 DIP protein Q00987 original sequence version: 145 DIP protein Q00987 original sequence version: 182 DIP protein Q00987 original sequence version: 151 DIP protein Q00987 original sequence version: 156 DIP protein Q00987-1 original sequence version: 160 DIP protein Q00987 original sequence version: 160 DIP protein Q00987 original sequence version: 168 DIP protein Q00987 original sequence version: 186 DIP protein Q00987 original sequence version: 206 F37CE163876BC983 mdm2-daxx 1 14 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey 15 unspecified role unspecified role bait bait association association Using a coimmunoprecipitation assay, the interaction between endogenous Daxx and Mdm2 was detected in several of the human cell lines examined, regardless of the status of p53 2 A and S5 E The interaction between Mdm2 and Daxx without p53 was shown in p53-negative HCT116 cells. Thus the interaction takes place regardless of p53 status. mdm2-daxx-1 11 15 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association The Daxx�Mdm2 interaction is likely to be direct, as shown by an in vitro pulldown assay with recombinant proteins 2 B daxx-usp7 5 14 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 13 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 physical association physical association 3 A HeLa S3 lysates were incubated with M2 beads (control) or M2 beads coated with recombinant Flag�Daxx. The bound proteins were resolved on SDS�PAGE and visualized by Coomassie blue staining. The 135 K protein band in the Flag�Daxx lane was identified as Hausp by mass spectrometry ubp7-daxx 1 12 unspecified role unspecified role prey prey 14 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey association association The interaction between endogenous Daxx and Hausp was verified using both a coimmunoprecipitation assay with anti-Daxx (Fig. 3b) and a reciprocal assay with anti-Hausp antibodies (see Supplementary Information, Fig. S2e). The Daxx� Hausp interaction was not dependent on either Mdm2 or p53, as it occurred in HCT116 (p53�/�) and Mdm2�/�p53�/� MEF cells (Fig. 3b). 3 B and S5g The interaction between all the 4 above proteins occurred in the p53 wild-type HCT116 cells and the interaction without p53 occurred in the HCY116 p53-negative cells. ubp7-daxx-1 7 14 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey physical association physical association The Daxx� Hausp interaction was not dependent on either Mdm2 or p53, as it occurred in HCT116 (p53�/�) and Mdm2�/�p53�/� MEF cells (Fig. 3b). 3 B and S5g ubp7-daxx-2 8 13 unspecified role unspecified role neutral component neutral component c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 14 unspecified role unspecified role neutral component neutral component n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range colocalization colocalization Immunofluorescence microscopy analysis revealed that Hausp and Daxx colocalized in nucleoplasma with accumulation in speckled structures 3 C ubp7-daxx-3 11 14 unspecified role unspecified role prey prey 13 unspecified role unspecified role bait bait direct interaction direct interaction Daxx binds directly to Hausp, as shown by an in vitro pulldown assay with purified recombinant proteins 3 D ubp7-daxx-4 10 15 unspecified role unspecified role bait bait 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 13 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 14 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 3 E and S5h A sequential immunoprecipitation assay showed that Daxx, Mdm2 and Hausp were present in the same complex ubp7-daxx-5 7 15 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 14 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association the Hausp�Mdm2 interaction was significantly enhanced by exogenous Daxx in a cotransfection assay 4 A Overexpression of Daxx enhances the interaction between Mdm2 and Daxx. ubp7-daxx-6 6 12 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey 13 unspecified role unspecified role bait bait 14 unspecified role unspecified role prey prey association association 4 B and S5i the Hausp�Mdm2 interaction was significantly enhanced by exogenous Daxx in a cotransfection assay (Fig. 4a), whereas this interaction was reduced in Daxx-deficient ES cells The Hausp-Mdm2 interaction was increased in the presence of Daxx. These results, in combination with the Mdm2- independent Daxx�Hausp interaction (Fig. 3b), suggested that Daxx is the central component of the Mdm2�Daxx�Hausp complex and physically connects Hausp to Mdm2 in vivo. ubp7-daxx-7 2 12 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey 13 unspecified role unspecified role bait bait 14 unspecified role unspecified role prey prey association association 4 C, 5 A (left panel), 5 B left panel the Hausp�Mdm2 interaction was significantly enhanced by exogenous Daxx in a cotransfection assay (Fig. 4a), whereas this interaction was reduced in Daxx-deficient ES cells (Fig. 4b) and Daxx siRNA-treated U2OS cells The interaction of endogenous Mdm2 and Hausp requires Daxx. These results, in combination with the Mdm2- independent Daxx�Hausp interaction (Fig. 3b), suggested that Daxx is the central component of the Mdm2�Daxx�Hausp complex and physically connects Hausp to Mdm2 in vivo. DNA damage caused by etoposide dissociates Daxx and Hausp from Mdm2. When the expression of ATM was decreased by siRNA, the interactions of Daxx with both Mdm2 and Hausp were less affected by etoposide. ubp7-daxx-8 2 15 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey 14 unspecified role unspecified role prey prey association association S2 A and S3 A Using a coimmunoprecipitation assay, the interaction between endogenous Daxx and Mdm2 was detected in several of the human cell lines examined, regardless of the status of p53 Hausp does not affect this interaction as show when U2OS cells were transfected with a Hausp-specific siRNA or a control siRNA. daxx-mdm2 3 15 unspecified role unspecified role neutral component neutral component 14 unspecified role unspecified role neutral component neutral component colocalization colocalization Endogenous Daxx and Mdm2 were mostly found in the nucleus. immunofluorescence microscopy analysis indicated that both endogenous Daxx and Mdm2 were found mainly in the nucleus, and their colocalization in subnuclear structures was evident when cells were treated with ALLN to stabilize Mdm2 S2B daxx-mdm2-1 10 15 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 14 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region contains second paired amphipathic helix domain (PAH2) required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association S2 D Deletion analyses revealed that the main binding domain for Mdm2 resides in a small region of Daxx (amino acids 157�260) Deletion analyses revealed that the main binding domain for Mdm2 resides in a small region of Daxx (amino acids 157�260) that encompasses the second paired amphipathic helix domain (PAH2). ubp7-daxx-9 2 13 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey 15 unspecified role unspecified role prey prey 14 unspecified role unspecified role prey prey association association S2 E and S4 C The interaction between endogenous Daxx and Hausp was verified using both a coimmunoprecipitation assay with anti-Daxx (Fig. 3b) and a reciprocal assay with anti-Hausp antibodies Etoposide stops Mdm2 from associating with the complex. Wortmannin prevents this disrupting effect. ubp7-daxx-10 4 14 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal contain the first paired amphipathic helix domain (PAH1) sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain c-terminal acid-rich region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 13 unspecified role unspecified role prey prey physical association physical association S2 F Deletion analyses revealed two regions in Daxx that associate with Hausp: the N-terminal 160 amino acids, which contain the first paired amphipathic helix domain (PAH1), and amino acids 347�570, which encompass the acid-rich region Two regions of Daxx associate with Hausp: the N-terminal 160 residues, containing the first paired amphipathis helix domain (PAH1) and residues 347-760, containing the acid-rich region. mdm2-daxx-2 2 15 unspecified role unspecified role prey prey 12 unspecified role unspecified role prey prey 14 unspecified role unspecified role bait bait association association S4 B The disruption of the binary Mdm2� Daxx interaction by DNA-damage signals seemed to be persistent and was not restored 4 h after treatment with etoposide (see Supplementary Information, Fig. S4b), whereas the binary Daxx�Hausp interaction was partially recovered by this time (see Supplementary Information, Fig. S4c). In contrast, the Daxx�p53 interaction was affected to a lesser extent Etoposide disrupts the Daxx-Mdm2 interaction and the Daxx-p53 interaction. usp7-hausp 9 14 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 15 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 13 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association Mutations of Mdm2 at Ser395 do not affect the interactions of Mdm2 with Daxx and Hausp. p53-/-Mdm2-/- MEF cells were co-transfected with HA-Daxx, HA-Hausp, together with Flag- Mdm2, -Mdm2(S395A), or -Mdm2(S395D) as indicated. Cells were treated with 20 μM MG-132 for 4h. Lysates and anti-Flag immunoprecipitates were analyzed by Western blot. S4 E Mutations of Mdm2 at Ser395 do not affect the interactions of Mdm2 with Daxx and Hausp.