UniProt UniProt http://www.uniprot.org Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. The Journal of biological chemistry 2003 imex curation Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu Accepted 2018-NOV-13 AT 13:44 GMT AT 13:44 GMT by PPORRAS The Journal of biological chemistry 2003 Only protein-protein interactions imex curation imex curation Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. The Journal of biological chemistry 2003 imex curation Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 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 predetermined predetermined participant predetermined Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu Accepted 2018-NOV-13 AT 13:44 GMT AT 13:44 GMT by PPORRAS The Journal of biological chemistry 2003 Only protein-protein interactions imex curation imex curation Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. The Journal of biological chemistry 2003 imex curation Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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 tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu Accepted 2018-NOV-13 AT 13:44 GMT AT 13:44 GMT by PPORRAS The Journal of biological chemistry 2003 Only protein-protein interactions imex curation imex curation Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. The Journal of biological chemistry 2003 imex curation Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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. fluorescence imaging fluorescence microscopy fluorescence imaging immunocytochemistry immunocytochemistry Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu Accepted 2018-NOV-13 AT 13:44 GMT AT 13:44 GMT by PPORRAS The Journal of biological chemistry 2003 Only protein-protein interactions imex curation imex curation Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. The Journal of biological chemistry 2003 imex curation Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu Accepted 2018-NOV-13 AT 13:44 GMT AT 13:44 GMT by PPORRAS The Journal of biological chemistry 2003 Only protein-protein interactions imex curation imex curation Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. Identification of a novel testicular orphan receptor-4 (TR4)-associated protein as repressor for the selective suppression of TR4-mediated transactivation. The Journal of biological chemistry 2003 imex curation Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast transcription compl transcriptional complementation assay transcription compl nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Yang Y., Wang X., Dong T., Kim E., Lin WJ., Chang C. chang@urmc.rochester.edu Accepted 2018-NOV-13 AT 13:44 GMT AT 13:44 GMT by PPORRAS Human testes cDNA library The Journal of biological chemistry 2003 Only protein-protein interactions imex curation imex curation nr2c2_human Nuclear receptor subfamily 2 group C member 2 NR2C2 TAK1 TR4 Orphan nuclear receptor TR4 Testicular receptor 4 Orphan nuclear receptor TAK1 protein protein human Homo sapiens Human MTSPSPRIQIISTDSAVASPQRIQIVTDQQTGQKIQIVTAVDASGSPKQQFILTSPDGAGTGKVILASPETSSAKQLIFTTSDNLVPGRIQIVTDSASVERLLGKTDVQRPQVVEYCVVCGDKASGRHYGAVSCEGCKGFFKRSVRKNLTYSCRSNQDCIINKHHRNRCQFCRLKKCLEMGMKMESVQSERKPFDVQREKPSNCAASTEKIYIRKDLRSPLIATPTFVADKDGARQTGLLDPGMLVNIQQPLIREDGTVLLATDSKAETSQGALGTLANVVTSLANLSESLNNGDTSEIQPEDQSASEITRAFDTLAKALNTTDSSSSPSLADGIDTSGGGSIHVISRDQSTPIIEVEGPLLSDTHVTFKLTMPSPMPEYLNVHYICESASRLLFLSMHWARSIPAFQALGQDCNTSLVRACWNELFTLGLAQCAQVMSLSTILAAIVNHLQNSIQEDKLSGDRIKQVMEHIWKLQEFCNSMAKLDIDGYEYAYLKAIVLFSPDHPGLTSTSQIEKFQEKAQMELQDYVQKTYSEDTYRLARILVRLPALRLMSSNITEELFFTGLIGNVSIDSIIPYILKMETAEYNGQITGASL DIP protein P49116 original sequence version: 79 DIP protein P49116 original sequence version: 97 5180BDC3F3C8BD79 nr2ca_human Nuclear receptor 2C2-associated protein NR2C2AP TR4 orphan receptor-associated 16 kDa protein TRA16 protein protein human Homo sapiens Human MTHSLVCPETVSRVSSVLNRNTRQFGKKHLFDQDEETCWNSDQGPSQWVTLEFPQLIRVSQLQIQFQGGFSSRRGCLEGSQGTQALHKIVDFYPEDNNSLQTFPIPAAEVDRLKVTFEDATDFFGRVVIYHLRVLGEKV D7BA1228FE9AA66E p49116-2 Nuclear receptor subfamily 2 group C member 2 NR2C2 TAK1 TR4 Orphan nuclear receptor TAK1 Orphan nuclear receptor TR4 Testicular receptor 4 protein protein human Homo sapiens Human MTSPSPRIQIISTDSAVASPQRIQGSEPASGPLSVFTSLNKEKIVTDQQTGQKIQIVTAVDASGSPKQQFILTSPDGAGTGKVILASPETSSAKQLIFTTSDNLVPGRIQIVTDSASVERLLGKTDVQRPQVVEYCVVCGDKASGRHYGAVSCEGCKGFFKRSVRKNLTYSCRSNQDCIINKHHRNRCQFCRLKKCLEMGMKMESVQSERKPFDVQREKPSNCAASTEKIYIRKDLRSPLIATPTFVADKDGARQTGLLDPGMLVNIQQPLIREDGTVLLATDSKAETSQGALGTLANVVTSLANLSESLNNGDTSEIQPEDQSASEITRAFDTLAKALNTTDSSSSPSLADGIDTSGGGSIHVISRDQSTPIIEVEGPLLSDTHVTFKLTMPSPMPEYLNVHYICESASRLLFLSMHWARSIPAFQALGQDCNTSLVRACWNELFTLGLAQCAQVMSLSTILAAIVNHLQNSIQEDKLSGDRIKQVMEHIWKLQEFCNSMAKLDIDGYEYAYLKAIVLFSPDHPGLTSTSQIEKFQEKAQMELQDYVQKTYSEDTYRLARILVRLPALRLMSSNITEELFFTGLIGNVSIDSIIPYILKMETAEYNGQITGASL 5D557E04602C1BAB nr2c2-nr2c2ap-1 6 8 unspecified role unspecified role unspecified role unspecified role over-expressed over expressed level over-expressed myristylation tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role unspecified role unspecified role over-expressed over expressed level over-expressed sos tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association nr2c2-nr2c2ap-2 4 8 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level colocalization colocalization Fig. 3A To further confirm that the interaction between TR4 and TRA16 in yeast cells may also occur in mammalian cells, we applied the immunocytofluorescence assay to test whether both TR4 and TRA16 are localized in the same cells. nr2c2ap-nr2c2-1 2 8 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region gal4 dna bd gal4 dna binding domain gal4 dna bd undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed region vp16 ad vp16 activation domain vp16 ad undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig. 3B Using the mammalian two-hybrid system to assay their direct interaction in COS-1 cells, we also found that the GAL4-TRA16 can interact strongly with VP16-TR4 (Fig. 3B,lane 4). nr2c2ap-nr2c2-2 1 8 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 9 unspecified role unspecified role prey prey partially purified partially purified region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro physical association physical association Fig. 3C We then used co-IP with in vitro translated TR4 and His tag fused TRA16 to assay their interaction. nr2c2ap-nr2c2-3 3 8 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 3D To further test that the endogenous TR4 could interact with the exogenous TRA16, the whole cell extracts from H1299 cells only transfected with TRA16 were immunoprecipitated with poly-clonal anti-His tag antibody, and the results show that TR4 and TRA16 can definitely co-immunoprecipitate (Fig. 3D) nr2c2-nr2c2-1 2 9 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region gal4 dna bd gal4 dna binding domain gal4 dna bd undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined lbd region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region vp16 ad vp16 activation domain vp16 ad undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 36 38 physical association physical association Fig. 9A, 9B We first demonstrated that TR4 can form the dimers via the interaction between TR4 and TR4-LBD (amino acids 224–615) in the mammalian two-hybrid assay as shown in Fig. 9A (lane 4). The addition of NR2C2AP (Q86WQ0) suppressed the interaction between TR4 and TR4-LBD nr2c2-nr2c2-2 2 9 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed region vp16 ad vp16 activation domain vp16 ad undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 8 inhibitor inhibitor ancillary ancillary over-expressed over expressed level over-expressed 9 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region gal4 dna bd gal4 dna binding domain gal4 dna bd undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined lbd region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Fig. 9A We first demonstrated that TR4 can form the dimers via the interaction between TR4 and TR4-LBD (amino acids 224–615) in the mammalian two-hybrid assay as shown in Fig. 9A (lane 4). The addition of NR2C2AP (Q86WQ0) suppressed the interaction between TR4 and TR4-LBD nr2c2ap-nr2c2-4 5 8 unspecified role unspecified role prey prey purified purified region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 9 unspecified role unspecified role bait bait partially purified partially purified region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain in vitro In vitro physical association physical association Fig. 9C In vitro co-IP experiments were performed to demonstrate that TRA16 could be co-immunoprecipitated with TR4DL but not TR4-N