MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Nucleic acids research 2008 imex curation Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-bmsc human Human bmsc Bone Marrow Stromal cells anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot 0tng 10tng 20tng Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com Accepted 2018-JUL-11 AT 08:32 BST AT 08:32 BST by LIVIAP Nucleic acids research 2008 imex curation Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Nucleic acids research 2008 imex curation Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com Accepted 2018-JUL-11 AT 08:32 BST AT 08:32 BST by LIVIAP Nucleic acids research 2008 imex curation Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Nucleic acids research 2008 imex curation Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com Accepted 2018-JUL-11 AT 08:32 BST AT 08:32 BST by LIVIAP Nucleic acids research 2008 imex curation Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Nucleic acids research 2008 imex curation Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-bmsc human Human bmsc Bone Marrow Stromal cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot 0tng 10tng 20tng Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com Accepted 2018-JUL-11 AT 08:32 BST AT 08:32 BST by LIVIAP Nucleic acids research 2008 imex curation Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells. Nucleic acids research 2008 imex curation Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-bmsc human Human bmsc Bone Marrow Stromal cells ch-ip chromatin immunoprecipitation assay ch-ip primer specific pcr primer specific pcr Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS., Lu C., Chen JQ., Zhou GP., Wu SH., Guan YF., Yuan CS. luchao8009@163.com Accepted 2018-JUL-11 AT 08:32 BST AT 08:32 BST by LIVIAP Nucleic acids research 2008 imex curation e2f1_human Transcription factor E2F1 Retinoblastoma-associated protein 1 Retinoblastoma-binding protein 3 PBR3 E2F1 RBBP3 pRB-binding protein E2F-1 protein protein human Homo sapiens Human MALAGAPAGGPCAPALEALLGAGALRLLDSSQIVIISAAQDASAPPAPTGPAAPAAGPCDPDLLLFATPQAPRPTPSAPRPALGRPPVKRRLDLETDHQYLAESSGPARGRGRHPGKGVKSPGEKSRYETSLNLTTKRFLELLSHSADGVVDLNWAAEVLKVQKRRIYDITNVLEGIQLIAKKSKNHIQWLGSHTTVGVGGRLEGLTQDLRQLQESEQQLDHLMNICTTQLRLLSEDTDSQRLAYVTCQDLRSIADPAEQMVMVIKAPPETQLQAVDSSENFQISLKSKQGPIDVFLCPEETVGGISPGKTPSQEVTSEEENRATDSATIVSPPPSSPPSSLTTDPSQSLLSLEQEPLLSRMGSLRAPVDEDRLSPLVAADSLLEHVREDFSGLLPEEFISLSPPHEALDYHFGLEEGEGIRDLFDCDFGDLTPLDF DIP protein Q01094 original sequence version: 173 DIP protein Q01094 original sequence version: 168 DIP protein Q01094 original sequence version: 149 DIP protein Q01094 original sequence version: 130 DIP protein Q01094 original sequence version: 125 DIP protein Q01094 original sequence version: 153 DIP protein Q01094 original sequence version: 180 003B3F654F0C60DF diablo_human_e2f1-site_dna-probe Human Diablo gene promoter transcription factor E2F1 binding BS3 site DNA probe dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid human Homo sapiens Human GCCCCGCGCGGAAGTT pawr_human PRKC apoptosis WT1 regulator protein Prostate apoptosis response 4 protein PAWR PAR4 protein protein human Homo sapiens Human MATGGYRTSSGLGGSTTDFLEEWKAKREKMRAKQNPPGPAPPGGGSSDAAGKPPAGALGTPAAAAANELNNNLPGGAPAAPAVPGPGGVNCAVGSAMLTRAAPGPRRSEDEPPAASASAAPPPQRDEEEPDGVPEKGKSSGPSARKGKGQIEKRKLREKRRSTGVVNIPAAECLDEYEDDEAGQKERKREDAITQQNTIQNEAVNLLDPGSSYLLQEPPRTVSGRYKSTTSVSEEDVSSRYSRTDRSGFPRYNRDANVSGTLVSSSTLEKKIEDLEKEVVRERQENLRLVRLMQDKEEMIGKLKEEIDLLNRDLDDIEDENEQLKQENKTLLKVVGQLTR DIP protein Q96IZ0 original sequence version: 31 DIP protein Q96IZ0 original sequence version: 110 7E7515455402DBF8 dbloh_human_gene Diablo homolog, mitochondrial DIABLO SMAC gene gene human Homo sapiens Human e2f1-1 5 6 unspecified role unspecified role prey prey subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role bait bait subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level -200 to -193bp sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f5 Chromatin immunoprecipitated with anti-Par-4 antibody or anti-E2F1 antibody was subjected to PCR using primers representing BS2 (−542 to −535 bp relative to ATG) site, another previously reported E2F1-binding site. No DNA could be amplified from the chromatins, (data not shown), demonstrating that Par-4 and E2F1 specifically bind to the BS3 (−200 to −193 bp relative to ATG) site on the Smac promoter. Chromatin immunoprecipitated with anti-Par-4 antibody or anti-E2F1 antibody was subjected to PCR using primers representing BS2 (−542 to −535 bp relative to ATG) site, another previously reported E2F1-binding site. No DNA could be amplified from the chromatins, (data not shown), demonstrating that Par-4 and E2F1 specifically bind to the BS3 (−200 to −193 bp relative to ATG) site on the Smac promoter. Glutamate diablo_human_e2f1_site_dna_probe-e2f1-3 2 7 unspecified role unspecified role bait bait purified purified biotin tag biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 6 unspecified role unspecified role prey prey partially purified partially purified human-bmsc human Human bmsc Bone Marrow Stromal cells physical association physical association f4b Indeed, we found that Par-4 and E2F1 existed in nuclear extracts from glutamate-treated cells (Figure 4A, lane 1; Figure 4B, lane 1). Indeed, we found that Par-4 and E2F1 existed in nuclear extracts from glutamate-treated cells (Figure 4A, lane 1; Figure 4B, lane 1). Glutamate diablo_human_e2f1_site_dna_probe-e2f1-1 2 7 unspecified role unspecified role bait bait purified purified biotin tag biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 8 unspecified role unspecified role prey prey partially purified partially purified human-bmsc human Human bmsc Bone Marrow Stromal cells physical association physical association f4a Indeed, we found that Par-4 and E2F1 existed in nuclear extracts from glutamate-treated cells (Figure 4A, lane 1; Figure 4B, lane 1). Indeed, we found that Par-4 and E2F1 existed in nuclear extracts from glutamate-treated cells (Figure 4A, lane 1; Figure 4B, lane 1). Glutamate diablo_human_e2f1_site_dna_probe-e2f1-2 3 6 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level human-bmsc human Human bmsc Bone Marrow Stromal cells 7 unspecified role unspecified role bait bait purified purified chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association f4c The neutralizing antibody against E2F1 caused disappearance of both the faster and the slower migrating complex, thus indicating the presence of E2F1 in both the complexes (Figure 4C, lane 5). These results supported that it was E2F1, not Par-4, in Par-4-E2F1 proteins complex that directly bound to Smac promoter, and that Par-4 might indirectly exert its transcriptional control on Smac gene though interacting with E2F1. The BS3 E2F1-binding site was also used as a probe to perform EMSA with nuclear extracts from the hBMSCs without treatment of glutamate. However, no DNA–protein complex was found (Figure 4C, lane 7). The neutralizing antibody against E2F1 caused disappearance of both the faster and the slower migrating complex, thus indicating the presence of E2F1 in both the complexes (Figure 4C, lane 5). These results supported that it was E2F1, not Par-4, in Par-4-E2F1 proteins complex that directly bound to Smac promoter, and that Par-4 might indirectly exert its transcriptional control on Smac gene though interacting with E2F1. The BS3 E2F1-binding site was also used as a probe to perform EMSA with nuclear extracts from the hBMSCs without treatment of glutamate. However, no DNA–protein complex was found (Figure 4C, lane 7). Glutamate e2f1-9 4 8 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level 6 unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3 As shown in Figure 3, Par-4 co-immunoprecipitated with E2F1 protein in nuclear extracts from the glutamate-treated hBMSCs, indicating the direct interaction between Par-4 and E2F1. By contrast, in nuclear extracts from the hBMSCs without the treatment of glutamate, Par-4 failed to associate with E2F1. As shown in Figure 3, Par-4 co-immunoprecipitated with E2F1 protein in nuclear extracts from the glutamate-treated hBMSCs, indicating the direct interaction between Par-4 and E2F1. By contrast, in nuclear extracts from the hBMSCs without the treatment of glutamate, Par-4 failed to associate with E2F1. Glutamate e2f1-10 1 8 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell myc tag myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region binding region region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role prey prey subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f6 As shown in Figure 3, Par-4 co-immunoprecipitated with E2F1 protein in nuclear extracts from the glutamate-treated hBMSCs, indicating the direct interaction between Par-4 and E2F1. By contrast, in nuclear extracts from the hBMSCs without the treatment of glutamate, Par-4 failed to associate with E2F1. As shown in Figure 3, Par-4 co-immunoprecipitated with E2F1 protein in nuclear extracts from the glutamate-treated hBMSCs, indicating the direct interaction between Par-4 and E2F1. By contrast, in nuclear extracts from the hBMSCs without the treatment of glutamate, Par-4 failed to associate with E2F1. diablo-lpar4-1 5 8 unspecified role unspecified role prey prey subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role bait bait subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level -200 to -193bp sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f5 Chromatin immunoprecipitated with anti-Par-4 antibody or anti-E2F1 antibody was subjected to PCR using primers representing BS2 (−542 to −535 bp relative to ATG) site, another previously reported E2F1-binding site. No DNA could be amplified from the chromatins, (data not shown), demonstrating that Par-4 and E2F1 specifically bind to the BS3 (−200 to −193 bp relative to ATG) site on the Smac promoter. Chromatin immunoprecipitated with anti-Par-4 antibody or anti-E2F1 antibody was subjected to PCR using primers representing BS2 (−542 to −535 bp relative to ATG) site, another previously reported E2F1-binding site. No DNA could be amplified from the chromatins, (data not shown), demonstrating that Par-4 and E2F1 specifically bind to the BS3 (−200 to −193 bp relative to ATG) site on the Smac promoter. Glutamate