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/ Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation Nfix regulates fetal-specific transcription in developing skeletal muscle. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell (0092-8674) 2010 imex curation 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 authors replied May 7th approx. Histone H1 = human aas 1-219. GST-Mef2A = human, both PKCtheta and Mef2A-flag are of human origin. HA-Nfix2 and YC155-Nfix2 are murine, pCMV.HAPax7 is murine Pax7. Tagged proteins identified by anti-tag antibodies. Additional detail of constructs supplied by the author. cossu.giulio@hsr.it Accepted 2010-JUN-20 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation pax7_mouse Paired box protein Pax-7 Pax7 Pax-7 protein protein mouse Mus musculus Mouse MAALPGAVPRMMRPGPGQNYPRTGFPLEVSTPLGQGRVNQLGGVFINGRPLPNHIRHKIVEMAHHGIRPCVISRQLRVSHGCVSKILCRYQETGSIRPGAIGGSKPRQVATPDVEKKIEEYKRENPGMFSWEIRDRLLKDGHCDRSTVPSVSSISRVLRIKFGKKEDDEEGDKKEEDGEKKAKHSIDGILGDKGNRLDEGSDVESEPDLPLKRKQRRSRTTFTAEQLEELEKAFERTHYPDIYTREELAQRTKLTEARVQVWFSNRRARWRKQAGANQLAAFNHLLPGGFPPTGMPTLPPYQLPDSTYPTTTISQDGGSTVHRPQPLPPSTMHQGGLAAAAAAADTSSAYGARHSFSSYSDSFMNPGAPSNHMNPVSNGLSPQVMSILSNPSAVPPQPQADFSISPLHGGLDSASSISASCSQRADSIKPGDSLPTSQSYCPPTYSTTGYSVDPVAGYQYSQYGQTAVDYLAKNVSLSTQRRMKLGEHSAVLGLLPVETGQAY 8ECC952C5C2FCA27 nfac4_mouse_gene Mouse Nuclear factor of activated T-cells, cytoplasmic 4 gene gene gene mouse Mus musculus Mouse 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. 4E89D0236A3F7235 mef2a_mouse Myocyte-specific enhancer factor 2A Mef2a protein protein mouse Mus musculus Mouse MGRKKIQITRIMDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSSNKLFQYASTDMDKVLLKYTEYNEPHESRTNSDIVETLRKKGLNGCESPDADDYFEHSPLSEDRFSKLNEDSDFIFKRGPPGLPPQNFSMSVTVPVTSPNALSYTNPGSSLVSPSLAASSTLADSSMLSPPPATLHRNVSPGAPQRPPSTGSASGMLSTTDLTVPNGAGNSPVGNGFVNSRASPNLIGNTGANSLGKVMPTKSPPPPGGGSLGMNSRKPDLRVVIPPSSKGMMPPLSEEEELELNAQRISSSQATQPLATPVVSVTTPSLPPQGLVYSAMPTAYNTDYSLTSADLSALQGFTSPGMLSLGQASAWQQHHLGQAALSSLVAGGQLSQGSNLSINTNQNINIKSEPISPPRDRMTPSGFQQQQQQPQQQPPPQPPQPQPRQEMGRSPVDSLSSSSSSYDGSDREDPRGDFHSPIVLGRPPNTEDRESPSVKRMRMDTWVT FE291C3E3E0A5E70 kpct_human Protein kinase C theta type PRKCQ nPKC-theta PRKCT protein protein human Homo sapiens Human MSPFLRIGLSNFDCGSCQSCQGEAVNPYCAVLVKEYVESENGQMYIQKKPTMYPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGKTEIWLELKPQGRMLMNARYFLEMSDTKDMNEFETEGFFALHQRRGAIKQAKVHHVKCHEFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIAKCTGSAINSRETMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQGLKCDACGMNVHHRCQTKVANLCGINQKLMAEALAMIESTQQARCLRDTEQIFREGPVEIGLPCSIKNEARPPCLPTPGKREPQGISWESPLDEVDKMCHLPEPELNKERPSLQIKLKIEDFILHKMLGKGSFGKVFLAEFKKTNQFFAIKALKKDVVLMDDDVECTMVEKRVLSLAWEHPFLTHMFCTFQTKENLFFVMEYLNGGDLMYHIQSCHKFDLSRATFYAAEIILGLQFLHSKGIVYRDLKLDNILLDKDGHIKIADFGMCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFGVLLYEMLIGQSPFHGQDEEELFHSIRMDNPFYPRWLEKEAKDLLVKLFVREPEKRLGVRGDIRQHPLFREINWEELERKEIDPPFRPKVKSPFDCSNFDKEFLNEKPRLSFADRALINSMDQNMFRNFSFMNPGMERLIS B3C53AB892D5210A kcrm_mouse_gene Mouse Creatine kinase M-type gene gene gene mouse Mus musculus Mouse Creatine kinase M-type nfix_mouse_gene Mouse Nuclear factor 1 X-type 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 362BA4FBCC792CE2 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 DIP protein Q02111 original sequence version: 120 7A16492116CD2880 nfix2-mef2a 1 11 unspecified role unspecified role prey prey 10 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 physical association physical association 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. The coimmunoprecipitations clearly revealed that Nfix2 is the only member of the Nfi family able to bind Mef2A nfix2-mef2a-1 5 15 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 10 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 physical association physical association 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-2 1 17 unspecified role unspecified role prey prey 11 unspecified role unspecified role prey prey 10 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 physical association physical association Fig 2E. Nfix2 was able to bind not only Mef2A but also PKCtheta. nfix2-mef2a-3 7 12 unspecified role unspecified role prey prey 15 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 10 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 physical association physical association 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 3 17 enzyme enzyme neutral component neutral component mouse-c2c12 Mouse C3H muscle myoblast c2c12 Mouse C3H muscle myoblast 10 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 15 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 phosphorylation phosphorylation reaction phosphorylation 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 2 10 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 17 unspecified role unspecified role bait bait physical association physical association 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. The amount of PKCθ and Nfix (by anti-HA antibody) in the immunoprecipitates was assessed by western blot (left). kcrm-mef2a 6 11 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey association association 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). This is a "positive control" reaction. enolase-nfix2 6 10 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 16 unspecified role unspecified role prey prey association association 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)". pax7-nfix2-1 4 14 unspecified role unspecified role prey prey 8 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 association association 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 4 9 unspecified role unspecified role prey prey 10 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 association association 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). kcrm-nfix2 6 13 unspecified role unspecified role prey prey 10 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 association association 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 6 11 unspecified role unspecified role bait bait 16 unspecified role unspecified role prey prey association association 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)".