ATF3 overexpression induces the binding activity of NF-κB and Nkx-2.5.

Representative EMSA blot of nuclear protein from cultured NRCM (A). ATF3 antibody (2 µl) caused supershift reaction (upper panel) and non-radiolabeled ATF3 probe (self) competes with radiolabeled ATF3 for binding to the probe, whereas mutated non-radiolabeled ATF3 probe (mut) had no effect (lower panel). EMSA of nuclear protein from ATF3 or LacZ adenovirus transduced (for 24 hours at the virus amount of 4 MOI) cultured cardiomyocytes (B–E). ATF3 (B), NF-κB (C), Nkx-2.5 (D) and AP-1 (E) binding activities are presented as bar graphs (mean ± SEM, n = 9–15 from at least 3 independent experiments) and representative blots. Also NF-κB (C), Nkx-2.5 (D) and AP-1 (E) antibodies (2 µl) cause supershift reactions (representative blots are shown). ET-1 stimulates ATF3 binding activity in cardiomyocytes (F). EMSA of nuclear protein from cultured cardiomyocytes treated with ET-1 (100 nM) for 1, 4, 12 and 24 hours. ATF3 binding activity is presented as bar graphs (mean ± SEM, n = 8 from 3 independent experiments) and representative blot. *P<0.05; **P<0.01; ***P<0.001.