Functional interaction of Nur77 with GATA4 in cardiomyocytes. (A) Myocytes were treated with ISO (10 μmol/liter) for 3 h. The nuclear fraction of cardiomyocytes was isolated and then subjected to immunoprecipitation with either normal IgG, anti-Nur77, or anti-GATA4 antibody. Immunocomplexes were immunoblotted with either anti-Nur77 or anti-GATA4 antibody. (B) Myocytes were transduced with either Ad-LacZ or Ad-DN-Nur77. At 48 h after transduction, cells were treated with ISO (10 μmol/liter) for 3 h. The nuclear fraction of cardiomyocytes was immunoprecipitated with either normal IgG or anti-Flag antibody and then immunoblotted with either anti-NFATc3, anti-GATA4, or anti-Flag antibody. (C) Myocytes were transduced with either Ad-LacZ or Ad-Nur77. At 48 h after transduction, nuclear extracts (NE) were isolated after ISO (10 µmol/liter) stimulation for 3 h and used for EMSA to detect the interaction of GATA4 with its conserved binding probe. A 100 times excess of unlabeled wild-type (WT) competitor was used to demonstrate the specificity of the shifted complex. To supershift the complex, 5 μl of GATA4 antibody was added to the binding reaction mixture. (D) Nur77 inhibits the binding of GATA4 to the BNP promoter. NRCMs were infected with Ad-Nur77 or Ad-LacZ for 48 h and then treated with vehicle or 10 μmol/liter ISO for 3 h. PCR analysis of sheared DNA from control and adenovirus-infected cells before immunoprecipitation (input) and after chromatin immunoprecipitation (ChIP) with antibody directed against GATA4 and quantitative analysis of ChIP results from 3 independent experiments are shown. *, P < 0.05 versus Ad-LacZ without ISO; #, P < 0.05 versus Ad-LacZ with ISO.