Fig. 3.

Effect of VWC2 on activin A-induced Smad2 phosphorylation. MC3T3-E1 cells (a) or mouse primary osteoblasts (b) were treated with VWC2 (200, 500, or 750 ng/ml indicated by +, ++, or +++, respectively) and activin A (250 ng/ml indicated by +) for 15 min and cell lysates were subjected to Western blot (WB) analysis with anti-phospho-Smad2 (pSmad2; upper panels in A, B) or anti-total Smad2 antibody (Smad2; lower panels in a, b). Experiments were performed at least three times and the representative results are shown. The intensities of the expression levels of phosphorylated Smad2 and total Smad2 were measured based on three experiments, the ratio of Smad2 phosphorylation was calculated, expressed as the mean + SD (left; MC3T3-E1 cells, right; mouse primary osteoblasts) and statistically analyzed. **P value < 0.01, *P value < 0.05