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/ A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J. Biol. Chem. (0021-9258) 2004 Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library orchard chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp Accepted 2007-SEP-31 by ORCHARD. 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library J. Biol. Chem. (0021-9258) 2004 orchard A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J. Biol. Chem. (0021-9258) 2004 Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library orchard 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp Accepted 2007-SEP-31 by ORCHARD. 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library J. Biol. Chem. (0021-9258) 2004 orchard A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J. Biol. Chem. (0021-9258) 2004 Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library orchard human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 confocal microscopy confocal microscopy antibody detection identification by antibody antibody detection Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis Please note it was not possible to determine the sequence of the huamn p21 oligonucleotide used in the gel shift assays mentioned in this paper. jfujita@virus.kyoto-u.ac.jp Accepted 2007-SEP-31 by ORCHARD. 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library J. Biol. Chem. (0021-9258) 2004 orchard A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J. Biol. Chem. (0021-9258) 2004 Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library orchard human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Sakurai T., Itoh K., Higashitsuji H., Nagao T., Nonoguchi K., Chiba T., Fujita J. Apoptosis - Interactions involving proteins with a function related to apoptosis jfujita@virus.kyoto-u.ac.jp Accepted 2007-SEP-31 by ORCHARD. 07-Sept-2007: Contacted by JYOTI. Human U-2 OS cDNA library J. Biol. Chem. (0021-9258) 2004 orchard zbt17_human Zinc finger and BTB domain-containing protein 17 ZBTB17 MIZ1 Zinc finger protein 60 Zinc finger protein 151 Myc-interacting zinc finger protein 1 ZNF151 ZNF60 protein protein human Homo sapiens Human MDFPQHSQHVLEQLNQQRQLGLLCDCTFVVDGVHFKAHKAVLAACSEYFKMLFVDQKDVVHLDISNAAGLGQVLEFMYTAKLSLSPENVDDVLAVATFLQMQDIITACHALKSLAEPATSPGGNAEALATEGGDKRAKEEKVATSTLSRLEQAGRSTPIGPSRDLKEERGGQAQSAASGAEQTEKADAPREPPPVELKPDPTSGMAAAEAEAALSESSEQEMEVEPARKGEEEQKEQEEQEEEGAGPAEVKEEGSQLENGEAPEENENEESAGTDSGQELGSEARGLRSGTYGDRTESKAYGSVIHKCEDCGKEFTHTGNFKRHIRIHTGEKPFSCRECSKAFSDPAACKAHEKTHSPLKPYGCEECGKSYRLISLLNLHKKRHSGEARYRCEDCGKLFTTSGNLKRHQLVHSGEKPYQCDYCGRSFSDPTSKMRHLETHDTDKEHKCPHCDKKFNQVGNLKAHLKIHIADGPLKCRECGKQFTTSGNLKRHLRIHSGEKPYVCIHCQRQFADPGALQRHVRIHTGEKPCQCVMCGKAFTQASSLIAHVRQHTGEKPYVCERCGKRFVQSSQLANHIRHHDNIRPHKCSVCSKAFVNVGDLSKHIIIHTGEKPYLCDKCGRGFNRVDNLRSHVKTVHQGKAGIKILEPEEGSEVSVVTVDDMVTLATEALAATAVTQLTVVPVGAAVTADETEVLKAEISKAVKQVQEEDPNTHILYACDSCGDKFLDANSLAQHVRIHTAQALVMFQTDADFYQQYGPGGTWPAGQVLQAGELVFRPRDGAEGQPALAETSPTAPECPPPAE DIP protein Q13105 original sequence version: 110 C159D177C8A2D4A3 maga4_human Melanoma-associated antigen 4 MAGE-4 antigen MAGE-X2 antigen MAGE-41 antigen Cancer/testis antigen 1.4 MAGEA4 MAGE4 protein protein human Homo sapiens Human MSSEQKSQHCKPEEGVEAQEEALGLVGAQAPTTEEQEAAVSSSSPLVPGTLEEVPAAESAGPPQSPQGASALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDELAHFLLRKYRAKELVTKAEMLERVIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPASNTYTLVTCLGLSYDGLLGNNQIFPKTGLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQENYLEYRQVPGSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGV C6AA2EE306963E7D mage_a4-miz_1 2 6 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 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 Miz-1 protein (Q13105) was identified from references provided in the text. 5A We identified 10 clones of 3.5 x 106 yeast clones transformed with a human U-2 OS cell cDNA library. Each clone proliferated on media containing the histidine inhibitor 3AT and was positive for {beta}-galactosidase staining (Fig. 5A and data not shown). DNA sequencing of the rescued plasmids revealed that two clones encoded the C-terminal 391 amino acids of Miz-1 mage_a4-miz_1-1 1 6 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 c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 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 physical association physical association Miz-1 protein (Q13105) was identified from references provided in the text. 5B To confirm that MAGE-A4{Delta}N1 interacts with Miz-1 in mammalian cells, COS-7 cells were co-transfected with plasmids expressing HA-tagged human full-length Miz-1 and FLAG-tagged MAGE-A4{Delta}N1. When cell lysates were immunoprecipitated with an anti-FLAG antibody, HA-Miz-1 was detected in the precipitates but not in those from cells co-transfected with the parental FLAG vector and HA-Miz-1 mage_a4-miz_1-2 1 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 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 5 cofactor cofactor coenzyme bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Miz-1 protein (Q13105) was identified from references provided in the text. 5B Reciprocally, MAGE-A4{Delta}N1 was detected in the anti-HA immunoprecipitates from cells co-transfected with plasmid expressing HA-Miz-1 and FLAG-MAGE-A4{Delta}N1 mage_a4-miz_1-3 3 6 unspecified role unspecified role neutral component neutral component c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role neutral component neutral component c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain colocalization colocalization Miz-1 protein (Q13105) was identified from references provided in the text. 5C Furthermore, in the HeLa cells expressing FLAG-MAGE-A4{Delta}N1 and HA-Miz-1, double immunofluorescence staining showed that expression of MAGE-A4{Delta}N1 did not affect the localization of Miz-1 and that both proteins were co-localized in the nucleus mage_a4-miz_1-4 4 5 unspecified role unspecified role prey prey 6 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 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 Miz-1 protein (Q13105) was identified from references provided in the text. 5E Interaction of MAGE-A4{Delta}N1 with endogenous Miz-1. HeLa cells were transfected with plasmids expressing FLAG-tagged MAGE-A4{Delta}N1 (+) or vector alone (-). Lysates (lower panel) and those immunoprecipitated with anti-FLAG antibody (upper panel) were analyzed by Western blotting using anti-Miz-1 antibody