BMP-2–mediated migration of hPASMCs requires activation of RhoA and Rac1. (A) Migration was assessed using the Boyden chamber assay as described in Materials and methods. Cells were starved for 48 h in starvation media (0.1% FBS) before seeding them in Boyden chambers placed in 24-well plates containing either starvation media alone or supplemented with 10 ng/ml BMP-2 or 20 ng/ml PDGF-BB. (B) RhoA (top) and Rac1 (bottom) activation was measured in lysates from cells stimulated with BMP-2 over a period of 6 h using the approach described in Materials and methods. Densitometry values are shown relative to total RhoA and Rac1 in whole cell lysates that were run in different gels. (C) Motility response to BMP-2 was assessed in cells incubated with either Y-27632 at 10, 50, and 100 mM or DMSO for 6 h. Bars represent means ± SEM from n = 3 different cell harvests in triplicate. ***, P < 0.0001 versus control or time 0; and ^##, P < 0.001 versus uninhibited control using one-way ANOVA with the Dunnett (A and B) or Bonferroni (C) tests. CON, control.