Effect of MEK/ERK inhibitor on GSK3β phosphorylation and GATA-4 translocation in neonatal rat cardiacmyocytes and H9c2 cells. H9c2 cells were exposed to 30 mmol/L glucose for 24 hrs, and subsequently the nuclear fraction and cytosolic fraction were isolated for Western blot analysis of GATA-4 expression and GSK3β phosphorylation (A and B). Changes in GATA-4 localization were also measured using a fluorescent microscope. Counter staining of the cell nucleus was achieved using DAPI (4', 6-diamidino-2-phenylindole) (A and D). H9c2 cells were incubated with PD98059 (20 mmol/L) for 30 min and subsequently exposed to the same glucose concentration (30 mmol/L) for 24 hr. The nuclear fractions were then isolated for Western blot analysis to measure phosphorylation of GSK3β (B). Result of western blot analysis in neonatal rat cardiomyocytes treated with HG at various doses(C). The results are presented as the mean ± SEM (n = 3 per group). *P < 0.05 as compared with the high glucose-treated group.