Figure 5.

GEFs of the Vav family mediate rapid activation of RhoG and Rac1. (A) HeLa cells overexpressing myc-SGEF were lysed without stimulation or after 30 s of EGF treatment (20 ng/ml). The fraction of active SGEF was precipitated using GST-RhoG-15A protein and revealed by Western blotting. (B) Knockdown efficiency of SGEF siRNA was tested by SGEF-specific RT-PCR from control- and SGEF-siRNA–transfected HeLa cells. RT-PCR of β2-microglobulin RNA served as a control. (C) HeLa cells were transfected 72 h before EGF stimulation with control or SGEF-specific siRNA. Rapid activation of RhoG or Rac1 after 30 s of EGF treatment was measured using ELMO- and PBD-pull-down assays, respectively. (D) Serum-starved HeLa cells were lysed without any stimulus or after 30 s of EGF treatment (20 ng/ml) and activated RhoG-specific GEFs were precipitated using GST-RhoG-15A protein. Immunoblots of the precipitates revealed that both Vav2 and Vav3 are rapidly activated in HeLa cells after EGF stimulation (right). (E and F) Experimental conditions as in B; siRNAs: control, Vav2, and Vav3 (E); control, Vav2/Vav3 simultaneously (F).