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/ Regulation of XIAP translation and induction by MDM2 following irradiation. Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell (1535-6108) 2009 imex curation Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer mzhou@emory.edu 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 2 human-eu-1 Homo sapiens EU-1 leukemia cell line eu-1 Human EU-1 leukemic cell line Established from pediatric ALL patients at Emory University School of Medicine, Atlanta, USA. Cultured EU-1 cells resemble primary leukemic cells: EU-1 cells have wt-p53 and overexpress MDM2. No Database ref available. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis mzhou@emory.edu Accepted 2010-MAR-15 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 2 imex curation Regulation of XIAP translation and induction by MDM2 following irradiation. Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell (1535-6108) 2009 imex curation Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer mzhou@emory.edu 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 2 in vitro In vitro nucl ac uv crosslink nucleic acid uv cross-linking assay nucl ac uv crosslink weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis An RNA binding Assay in which the protein/RNA complexes were UV crosslinked, run on an SDS-PAGE gel, and imaged by autoradiography. mzhou@emory.edu Accepted 2010-MAR-15 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 2 imex curation Regulation of XIAP translation and induction by MDM2 following irradiation. Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell (1535-6108) 2009 imex curation Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer mzhou@emory.edu 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 2 human-sh_ep Homo sapiens SH-EP: human neuroblastoma cell line sh_ep SH-EP human neuroblastoma cell line This is one of two morphologically distinct cell types resulting from the subcloning of the SK-N-SH NB cell line: SH-SY5Y (N-type) and SH-EP (S-type); SH-EP (SHEP) is an Epithelial-like clone. (The first reference to it I found was in 1978. There are no Cabri or ATCC reference numbers.) nucl ac uv crosslink nucleic acid uv cross-linking assay nucl ac uv crosslink weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Cell extracts from SH-EP1 were incubated with probes, UV crosslinked, and then immunoprecipitated with antibodies. mzhou@emory.edu Accepted 2010-MAR-15 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 2 imex curation Regulation of XIAP translation and induction by MDM2 following irradiation. Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell (1535-6108) 2009 imex curation Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer mzhou@emory.edu 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 2 human-sh_ep Homo sapiens SH-EP: human neuroblastoma cell line sh_ep SH-EP human neuroblastoma cell line This is one of two morphologically distinct cell types resulting from the subcloning of the SK-N-SH NB cell line: SH-SY5Y (N-type) and SH-EP (S-type); SH-EP (SHEP) is an Epithelial-like clone. (The first reference to it I found was in 1978. There are no Cabri or ATCC reference numbers.) anti bait coip anti bait coimmunoprecipitation anti bait coip primer specific pcr primer specific pcr Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Following coimmunoprecipitation with anti-MDM2, the XIAP mRNA was detected by RT-PCR analysis. mzhou@emory.edu Accepted 2010-MAR-15 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 2 imex curation Regulation of XIAP translation and induction by MDM2 following irradiation. Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell (1535-6108) 2009 imex curation Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer mzhou@emory.edu 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 2 in vitro In vitro nucl ac uv crosslink nucleic acid uv cross-linking assay nucl ac uv crosslink weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gu L., Zhu N., Zhang H., Durden DL., Feng Y., Zhou M. Apoptosis - Interactions involving proteins with a function related to apoptosis I left this as a separate Experiment, though it is the same Interaction Detection and Part Detact as gu-2009-3, as the Exp- modification is different (slightly).(I also altered the nucleci acid as I could enter the sequence for this ). Antisense mapping of the XIAP IRES/MDM2 binding site. Antisense oligonucleotides were annealed to probe 1, then incubated with full-length MDM2, UV crosslinked, run on an SDS-PAGE gel, and imaged by autoradiography. mzhou@emory.edu Accepted 2010-MAR-15 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 2 imex curation 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 xiap_mrna_ires Human XIAP mRNA IRES rna ribonucleic acid RNA rna human Homo sapiens Human A nucleotide sequence of the long 5'UTR of XIAP mRNA containing a 162-bp IRES, (reported by Holcic et al 1999) and submitted by Livingston et al. (AY886519). Sequence available but could not be copied - nor could I locate the stated DNA sequence to the stated reference. xiap_ires_fragment Human XIAP mRNA IRES fragment rna ribonucleic acid RNA rna human Homo sapiens Human GGAUUUCCUAAUAUAAUGUUCUCUUUUU F4CF84A68B09A82F 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 xiap_mrna Human XIAP mRNA rna ribonucleic acid RNA rna human Homo sapiens Human A genomic DNA reference was originally given (in pmid19411066):AY886519, from which the mRNA link below was derived. An RNA reference was also given - NM001167 - which does not now exist in Genbank. mdm2-p53 1 9 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait physical association physical association MDM2 dissociated from p53 after IR treatment. Cells were treated with 10 Gy Infra-red radiation Fig 1B. mdm2-p53-3 1 9 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association MDM2 dissociated from p53 after IR treatment. Fig 1B. Cells were treated with 10 Gy Infra-red radiation mdm2-p53-4 4 6 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association Fig 4B. MDM2 protein was able to bind XIAP mRNA. mdm2-xiap 2 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association Fig 4C. UV crosslinking and gel electrophoresis showed that the MDM2 protein strongly bound to the XIAP IRES RNA in vitro. UV light, using a 254-nm UV light source set at 400,000uJ/square cm. The RNA probe was radiolabeled with 32P. mdm2-xiap-3 3 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association Fig 4D. UV light, using a 254-nm UV light source set at 400,000uJ/square cm. Binding of MDM2 to XIAP IRES was further confirmed by similar UV crosslinking and gel electrophoresis using cellular extract of SH-EP1 cells and IP with anti-MDM2 antibody. The RNA probe was radiolabeled with 32P. mdm2-xiap-2 2 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 c-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain c-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Q00987:p.Gly448Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain G S Wed May 02 15:04:37 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly448ser' Tue Jun 30 19:14:27 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly448Ser' ecobb Escherichia coli (strain B / BL21) physical association physical association Fig 5A, 5B. UV light, using a 254-nm UV light source set at 400,000uJ/square cm. We performed UV crosslinking and gel electrophoresis to screen for the domain of MDM2 required for XIAP IRES binding. The binding of fragment (434�491) to the XIAP IRES was greatly reduced, suggesting that 10 amino acids (425�434) are critical for proper binding. In addition, when the last 11 hydrophobic amino acids were deleted, the binding of this fragment (425�480) to XIAP IRES was abolished. The G448S mutation greatly reduced the specific XIAP IRES binding of MDM2. The RNA probe was radiolabeled with 32P. mdm2-xiap-1 5 8 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Fig 5D, 5E. UV light, using a 254-nm UV light source set at 400,000uJ/square cm. We also wished to determine what region on the XIAP IRES is critical for MDM2 binding. We observed that antisense A8 containing the critical polypyrimidine tract that is essential for XIAP IRES function. Antisense A7 , to a lesser extent, inhibited the binding of MDM2 to XIAP IRES. A combination of A8 and A7 completely blocked the binding between MDM2 and XIAP IRES. The RNA probe was radiolabeled with 32P.