Fig. 3. Effect of RhoA silencing on cell spreading. (A) An siRNA targeting RhoA strongly reduced the RhoA protein level without a significant effect on Rac1 and Cdc42 protein levels. Western blot analysis of whole cell lysates of VSMCs transfected with 20 nM of a control siRNA (Control) or an siRNA targeting RhoA or left untransfected (C). 48 hours post-transfection, cells were lysed in SDS-PAGE loading buffer and analyzed by immunoblotting with specific antibodies to RhoA, Rac1, Cdc42 and p42 MAP kinase. (B) Inhibition of RhoA with an siRNA stabilizes process formation. Representative phase-contrast microscopy of quiescent VSMCs allowed to spread for 4 hours (a-d) on tissue culture dishes coated with laminin and either Fc (a,b) or A1 (c,d). VSMCs transfected with control siRNA (a,c) or an siRNA targeting RhoA (b,d) were used 48 hours post-transfection. Bar, 20 µm.