Figure 2

Activation of Fer and non-oncogenic Src. (A) RhoGDIα was expressed alone or with c-Src as indicated above the panel. Cells in lanes marked with a "+" were stimulated with H₂O₂ (10 mM) for 15 minutes and RhoGDIα was immunoprecipitated. Anti-phosphotyrosine (pY20) blots were stripped and re-probed with RhoGDIα antibodies (Santa Cruz). (B) Mouse embryonic fibroblasts (MEFs), mouse endothelial cells and cells transfected with Fer were treated with 5 mM of H₂O₂ for the indicated time. Fer protein was immunoprecipitated from the lysates followed by a pY20 immunoblot to detect activated tyrosine phosphorylated Fer (Abcam). (C) COS-1 cells transfected with Xpress-tagged RhoGDIα were stimulated or not with H₂O₂, as indicated below the panels. RhoGDIα antibodies (Upstate Biotechnology) were used for immunoprecipitation. Antibodies used for Western blotting are indicated to the left of the panels. Fer, CH-6 antibodies. This result is representative of two independent experiments.