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/ http://www.ebi.ac.uk/ messina-2010-4 Nfix regulates fetal-specific transcription in developing skeletal muscle. mouse-fetal-myotubes Mus musculus fetal myotubes foetal_myotube Foetal myotube Myotubes taken from a fetal as opposed to a embryonic or adult source. Foetus is defined as the stage at which tissue diferentiation is initiated. Muscle fibers form from the fusion of myoblasts (muscle stem cells) into multi-nucleated fibers (myotubes). ch-ip chromatin immunoprecipitation assay ch-ip nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation messina-2010-1 Nfix regulates fetal-specific transcription in developing skeletal muscle. mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation messina-2010-7 Nfix regulates fetal-specific transcription in developing skeletal muscle. mouse-fetal-myoblasts Mus musculus fetal myoblasts foetal_myoblast Foetal myoblast-muscle stem cell Muscle fibers form from the fusion of myoblasts (muscle stem cells) into multi-nucleated fibers (called myotubes). ch-ip chromatin immunoprecipitation assay ch-ip nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation messina-2010-2 Nfix regulates fetal-specific transcription in developing skeletal muscle. mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation messina-2010-3 Nfix regulates fetal-specific transcription in developing skeletal muscle. human-jeg3 Homo sapiens choriocarcinoma JEG3 jeg3 Human choriocarcinoma JEG3 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Tagged proteins identified by anti-tag antibodies. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation messina-2010-5 Nfix regulates fetal-specific transcription in developing skeletal muscle. in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation messina-2010-6 Nfix regulates fetal-specific transcription in developing skeletal muscle. mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Messina G., Biressi S., Monteverde S., Magli A., Cassano M., Perani L., Roncaglia E., Tagliafico E., Starnes L., Campbell CE., Grossi M., Goldhamer DJ., Gronostajski RM., Cossu G. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Cell (0092-8674) 2010 Only protein-protein interactions imex curation mef2a_mouse Myocyte-specific enhancer factor 2A Mef2a protein protein mouse Mus musculus Mouse MGRKKIQITRIMDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSSNKLFQYASTDMDKVLLKYTEYNEPHESRTNSDIVETLRKKGLNGCESPDADDYFEHSPLSEDRFSKLNEDSDFIFKRGPPGLPPQNFSMSVTVPVTSPNALSYTNPGSSLVSPSLAASSTLADSSMLSPPPATLHRNVSPGAPQRPPSTGSASGMLSTTDLTVPNGAGNSPVGNGFVNSRASPNLIGNTGANSLGKVMPTKSPPPPGGGSLGMNSRKPDLRVVIPPSSKGMMPPLSEEEELELNAQRISSSQATQPLATPVVSVTTPSLPPQGLVYSAMPTAYNTDYSLTSADLSALQGFTSPGMLSLGQASAWQQHHLGQAALSSLVAGGQLSQGSNLSINTNQNINIKSEPISPPRDRMTPSGFQQQQQQPQQQPPPQPPQPQPRQEMGRSPVDSLSSSSSSYDGSDREDPRGDFHSPIVLGRPPNTEDRESPSVKRMRMDTWVT kcrm_mouse_gene Mouse Creatine kinase M-type gene gene gene mouse Mus musculus Mouse Creatine kinase M-type p70257-2 Nuclear factor 1 X-type Nfix CCAAT-box-binding transcription factor Nuclear factor I/X TGGCA-binding protein protein protein mouse Mus musculus Mouse MYSPYCLTQDEFHPFIEALLPHVRAFSYTWFNLQARKRKYFKKHEKRMSKDEERAVKDELLGEKPEIKQKWASRLLAKLRKDIRPEFREDFVLTITGKKPPCCVLSNPDQKGKIRRIDCLRQADKVWRLDLVMVILFKGIPLESTDGERLYKSPQCSNPGLCVQPHHIGVTIKELDLYLAYFVHTPESGQSDSSNQQGDADIKPLPNGHLSFQDCFVTSGVWNVTELVRVSQTPVATASGPNFSLADLESPSYYNINQVTLGRRSITSPPSTSSTKRPKSIDDSEMESPVDDVFYPGTGRSPAAGSSQSSGWPNDVDAGSPRATASALHFPSTSIIQQSSPYFTHPTIRYHHHHGQDSLKEFVQFVCSDGSGQATGQHSQRQAPPLPTGLSASDPGTATF Contains a phosphoserine at position 301. Contains a phosphoserine at position 320. enob_mouse_gene Mouse Beta-enolase gene gene gene mouse Mus musculus Mouse kpct_mouse Protein kinase C theta type Prkcq Pkcq nPKC-theta protein protein mouse Mus musculus Mouse MSPFLRIGLSNFDCGTCQACQGEAVNPYCAVLVKEYVESENGQMYIQKKPTMYPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGRTEIWLELKPQGRMLMNARYFLEMSDTKDMSEFENEGFFALHQRRGAIKQAKVHHVKCHEFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIAKCTGSAINSRETMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQGLKCDACGMNVHHRCQTKVANLCGINQKLMAEALAMIESTQQARSLRDSEHIFREGPVEIGLPCSTKNETRPPCVPTPGKREPQGISWDSPLDGSNKSAGPPEPEVSMRRTSLQLKLKIDDFILHKMLGKGSFGKVFLAEFKRTNQFFAIKALKKDVVLMDDDVECTMVEKRVLSLAWEHPFLTHMFCTFQTKENLFFVMEYLNGGDLMYHIQSCHKFDLSRATFYAAEVILGLQFLHSKGIVYRDLKLDNILLDRDGHIKIADFGMCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFGVLVYEMLIGQSPFHGQDEEELFHSIRMDNPFYPRWLEREAKDLLVKLFVREPEKRLGVRGDIRQHPLFREINWEELERKEIDPPFRPKVKSPYDCSNFDKEFLSEKPRLSFADRALINSMDQNMFSNFSFINPGMETLICS nfix_mouse_gene Mouse Nuclear factor 1 X-type gene gene gene mouse Mus musculus Mouse pax7_mouse Paired box protein Pax-7 Pax7 Pax-7 protein protein mouse Mus musculus Mouse MAALPGAVPRMMRPGPGQNYPRTGFPLEVSTPLGQGRVNQLGGVFINGRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPRQVATPDVEKKIEEYKRENPGMFSWEIRDRLLKDGHCDRSTVPSVSSISRVLRIKFGKKEDDEEGDKKEEDGEKKAKHSIDGILGDKGNRLDEGSDVESEPDLPLKRKQRRSRTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQLAAFNHLLPGGFPPTGMPTLPPYQLPDSTYPTTTISQDGGSTVHRPQPLPPSTMHQGGLAAAAAAADTSSAYGARHSFSSYSDSFMNPGAPSNHMNPVSNGLSPQVMSILSNPSAVPPQPQADFSISPLHGGLDSASSISASCSQRADSIKPGDSLPTSQSYCPPTYSTTGYSVDPVAGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGEHSAVLGLLPVETGQAY nfac4_mouse_gene Mouse Nuclear factor of activated T-cells, cytoplasmic 4 gene gene gene mouse Mus musculus Mouse mef2a_human Myocyte-specific enhancer factor 2A MEF2A MEF2 Serum response factor-like protein 1 protein protein human Homo sapiens Human MGRKKIQITRIMDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSSNKLFQYASTDMDKVLLKYTEYNEPHESRTNSDIVEALNKKEHRGCDSPDPDTSYVLTPHTEEKYKKINEEFDNMMRNHKIAPGLPPQNFSMSVTVPVTSPNALSYTNPGSSLVSPSLAASSTLTDSSMLSPPQTTLHRNVSPGAPQRPPSTGNAGGMLSTTDLTVPNGAGSSPVGNGFVNSRASPNLIGATGANSLGKVMPTKSPPPPGGGNLGMNSRKPDLRVVIPPSSKGMMPPLSEEEELELNTQRISSSQATQPLATPVVSVTTPSLPPQGLVYSAMPTAYNTDYSLTSADLSALQGFNSPGMLSLGQVSAWQQHHLGQAALSSLVAGGQLSQGSNLSINTNQNISIKSEPISPPRDRMTPSGFQQQQQQQQQQQPPPPPQPQPQPPQPQPRQEMGRSPVDSLSSSSSSYDGSDREDPRGDFHSPIVLGRPPNTEDRESPSVKRMRMDAWVT kpct_human Protein kinase C theta type PRKCQ nPKC-theta PRKCT protein protein human Homo sapiens Human MSPFLRIGLSNFDCGSCQSCQGEAVNPYCAVLVKEYVESENGQMYIQKKPTMYPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGKTEIWLELKPQGRMLMNARYFLEMSDTKDMNEFETEGFFALHQRRGAIKQAKVHHVKCHEFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIAKCTGSAINSRETMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQGLKCDACGMNVHHRCQTKVANLCGINQKLMAEALAMIESTQQARCLRDTEQIFREGPVEIGLPCSIKNEARPPCLPTPGKREPQGISWESPLDEVDKMCHLPEPELNKERPSLQIKLKIEDFILHKMLGKGSFGKVFLAEFKKTNQFFAIKALKKDVVLMDDDVECTMVEKRVLSLAWEHPFLTHMFCTFQTKENLFFVMEYLNGGDLMYHIQSCHKFDLSRATFYAAEIILGLQFLHSKGIVYRDLKLDNILLDKDGHIKIADFGMCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFGVLLYEMLIGQSPFHGQDEEELFHSIRMDNPFYPRWLEKEAKDLLVKLFVREPEKRLGVRGDIRQHPLFREINWEELERKEIDPPFRPKVKSPFDCSNFDKEFLNEKPRLSFADRALINSMDQNMFRNFSFMNPGMERLIS kcrm-mef2a 896655 n/a 896662 unspecified role unspecified role bait bait n/a 896663 unspecified role unspecified role prey prey association association false false false Fig S2C. ChIP assay performed on HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed the direct binding of Nfix on MCK and beta-enolase enhancer/promoter. Binding to the MCK enhancer/promoter regions was found at: -1200/-1048 and -2500/-2400. Fig S2C image legend states: Unpurified fetal myoblasts (E16.5) were (used)... However, Text states: " HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed.... (Figure S2C)" (were used). enolase-nfix2 896655 n/a 896664 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 896665 unspecified role unspecified role prey prey association association false false false Fig S2C. ChIP assay performed on HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed the direct binding of Nfix on MCK and beta-enolase enhancer/promoter. Binding to the Beta-enolase enhancer/promoter regions was found at: region +504/+637. Fig S2C image legend states: Unpurified fetal myoblasts (E16.5) were (used)... However, Text states: " HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed.... (Figure S2C) (were used)". kcrm-nfix2 896655 n/a 896663 unspecified role unspecified role prey prey n/a 896664 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false Fig S2C. ChIP assay performed on HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed the direct binding of Nfix on MCK and beta-enolase enhancer/promoter. Binding to the MCK enhancer/promoter regions was found at: -1200/-1048 and -2500/-2400. Fig S2C image legend states: Unpurified fetal myoblasts (E16.5) were (used)... However, Text states: " HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed.... (Figure S2C) (were used)". enolase-mef2a 896655 n/a 896662 unspecified role unspecified role bait bait n/a 896665 unspecified role unspecified role prey prey association association false false false Fig S2C. ChIP assay performed on HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed the direct binding of Nfix on MCK and beta-enolase enhancer/promoter. Binding to the Beta-enolase enhancer/promoter regions was found at: region +504/+637. Fig S2C image legend states: Unpurified fetal myoblasts (E16.5) were (used)... However, Text states: " HA-Nfix2-expressing fetal myotubes (E16.5 HA-Nfix2) confirmed.... (Figure S2C) (were used)". nfix2-mef2a 896656 n/a 896662 unspecified role unspecified role prey prey n/a 896664 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Fig 2B. Nfi is able to specifically bind Mef2A but not Mef2C. Further coimmunoprecipitations clearly revealed that Nfix2 is the only member of the Nfi family able to bind Mef2A. nfix2-mef2a-2 896656 n/a 896666 unspecified role unspecified role prey prey n/a 896662 unspecified role unspecified role prey prey n/a 896664 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Fig 2E. pax7-nfix2-1 896657 n/a 896667 unspecified role unspecified role prey prey n/a 896668 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false Fig 7C. Pax7 directly binds, with different affinity, to four putative Pax7 binding sites on the Nfix promoter, regions: -1677/-1659, -2290/-2272, -3898/-3880, and -4274/-4256. nfix2-nfatc4-1 896657 n/a 896669 unspecified role unspecified role prey prey n/a 896664 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false Fig 6C. A ChIP assay for Nfix2 failed to detect direct binding on the slow MyHC promoter, however direct binding of Nfix2 on the promoter of NFATc4 was demonstrated at (-3770/-3750). nfix2-mef2a-1 896658 n/a 896670 unspecified role unspecified role neutral component neutral component region n-terminus of YFP tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 896664 unspecified role unspecified role neutral component neutral component region c-terminus of YFP tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false physical association physical association false false false Fig 2D. This further confirms that Nfix2 is the only member of the Nfi family able to bind Mef2A was obtained by BIFC using Nfix2 and Mef2A fused to the amino(N)- or carboxy(C)-terminal of the yellow fluorescent protein (YFP), respectively, and transfected into C2C12 myoblasts. nfix2-mef2a-3 896659 n/a 896671 unspecified role unspecified role prey prey n/a 896670 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 false n/a 896664 unspecified role unspecified role ancillary ancillary region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false Fig 2F. Mef2A and PKCtheta were not able to bind directly to each other, while binding was rescued in the presence of Nfix2, suggesting that Nfix acts as a bridge between Mef2A and PKCtheta. nfix2-mef2a-4 896660 n/a 896666 enzyme enzyme neutral component neutral component mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast n/a 896664 unspecified role unspecified role ancillary ancillary region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 896670 enzyme target enzyme target substrate substrate neutral component neutral component 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 false phosphorylation phosphorylation reaction phosphorylation false false false Fig 2G. Myoblast PKCtheta was able to weakly phosphorylate Mef2A, but this ability was strongly enhanced in the presence of Nfix2. phosphorylation of Mef2a. pkc-mef2a-5 896661 n/a 896664 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 false n/a 896666 unspecified role unspecified role bait bait physical association physical association false false false Fig 2G LHS. PKCtheta was immunoprecipitated in C2C12 myoblasts transfected with HA-Nfix2 plasmid. The amount of PKCθ and Nfix (by anti-HA antibody) in the immunoprecipitates was assessed by western blot.