Fig. 3. Knockdown of Lsc/p115 RhoGEF and LARG decreases formation of stress fibers and focal adhesions and RhoA activity downstream of FN. (A) REF52 cells were transfected with either control or siRNA oligonucleotides against Lsc and LARG as described in Materials and Methods. 72 hours post transfection, the cells were serum starved, held in suspension for 2 hours and plated onto FN-coated coverslips for 90 minutes. The cells were fixed and stained with phalloidin and antibody against phosphotyrosine to visualize stress fibers and focal adhesions. Bar, 40 µm. (B) Control cells or cells transfected with siRNA against Lsc and LARG were lysed and samples blotted with antibodies against Lsc and LARG to demonstrate the efficiency of knockdown. Identical blots with an antibody against Lfc show that protein levels of the closely related GEF Lfc are unaffected, demonstrating the specificity of the knockdown. (C) Fibroblasts were transfected with either control or siRNA oligonucleotides against Lsc and LARG. 72 hours post transfection, the cells were serum-starved, held in suspension for 2 hours and plated onto FN-coated dishes for 60 minutes. The cells were then lysed, GST-RBD pulldowns performed and samples blotted with an antibody against RhoA to visualize the levels of RhoA activity. (D) Fibroblasts were transfected with siRNA oligonucleotides against Lsc and LARG. 48 hours post transfection, the cells were re-transfected with either a vector control or a V5-tagged full-length p115 RhoGEF construct. 72 hours post transfection, the cells were processed for Rho activity assays, as described in (C) above. Lysates were also blotted with an antibody against V5 to show the expression levels of V5-p115(FL).