Fig. 3. Effect of depletion of GEFs on EGF-induced Cdc42 activation. (A) Cells were prepared as described for Figs 1 and 2, except that pRaichu-Cdc42 was transfected instead of pRaichu-Rac1. After serum starvation, cells were stimulated with 25 ng/ml EGF and examined for Cdc42 activity by FRET microscopy as described in the text. The averages of the fold increase over the control cells are shown + s.d. Numbers of cells examined for each condition were as follows: control siRNA, n=23; Vav2 KD, n=6; Tiam1 KD, n=13; control shRNA, n=6; Asef KD, n=3. ^*P<0.05, significant difference as compared with the control by t-test analysis (Student's t-test). (B) Control and knockdown cells were starved for 6-12 h, stimulated with 25 ng/ml EGF for 2 minutes or left unstimulated, and were examined for active Cdc42 by pull-down assay. Experiments were repeated at least three times, and average values of the fold increase over the mock-treated cells are shown + s.d. ^*P<0.05, significant difference as compared with the control by t-test analysis (Student's t-test).