Fig. 4.

ERK2, but not ERK1, is involved in BMP-4-induced Smad1/5 activation in MG63 cells. A, MG63 cells were kept as controls (C) or treated with BMP-4 (25 ng/ml) for 10 min (10′), 30 min (30′), and 1 h, and their ERK phosphorylation was determined by Western blot analysis. Data are mean ± sem from three independent experiments and are presented as percentage changes in band density from control cells normalized to ERK2 protein levels. *, P < 0.05 vs. unstimulated control cells. B–E, MG63 cells were transfected with control siRNA (siCL), specific siRNA of BMPRIA (siRIA) and BMPRIB (siRIB) alone or simultaneously (siRIA/B) (B), or ERK1 (siERK1) and ERK2 (siERK2) (E) (40 nm for each) for 48 h. In some experiments, MG63 cells were treated with PD98059 (PD; 30 μm) for 1 h (C) or transfected with control empty vector pcDNA3 or an expression plasmid encoding MEK1 (D) (1 μg) for 48 h. The cells were then kept as controls (C) or treated with BMP-4 (B) for 10 min. The phosphorylations of ERK (B and D) or Smad1/5 (C–E) were determined by Western blot analysis. Results are representative of three independent experiments with similar results. DMSO, Dimethylsulfoxide.