Figure 4.

Src negatively regulates RhoA activity in C8161.9 cells. A, C) C8161.9 cells were left untreated, treated with SU6656 (SU), or B, D) infected with no virus (control), wild type Src (WT Src), or dominant interfering mutant Src (SrcK295M) virus. Cells were then placed in suspension (S) or plated on vitronectin (VN) for 45 minutes. A, B) the amount of GTP-bound RhoA (RhoAGTP) was monitored using GST-RBD pull-down assays (RBD IP) and immunoblotting. Total levels of RhoA and over expressed Src in the cell extracts were monitored by immunoblotting (RhoA Blot, Src Blot). Extent of RhoA activation was normalized to total RhoA for each sample. Data were expressed as the fold in increase in RhoA activation compared to control or untreated samples. C, D) The level of p190RhoGAP tyrosine phosphorylation was measured by immunoblotting of immunoprecipitates (p190GAP IP) with anti-phosphotyrosine antibodies (P-Tyr Blot). Total levels of p190RhoGAP in the immunoprecipitates were measured by immunoblotting with anti-p190RhoGAP antibodies (p190GAP Blot).