Fig. 2. Effect of Asef deficiency on EGF-induced activation of Rac1. (A) A431 cells were transfected with pSuper-Luc or pSuper-Asef. After selection with puromycin, the expression level of Asef mRNA was examined by RT-PCR (left). (B) To confirm the the efficiency of knockdown, cells transfected with a control shRNA expression vector and an Asef expression vector were analyzed by immunoblotting (right). Signal intensities of each band were quantified and are shown below each band as a relative strength to the control (in %). (C) A431 cells were transfected with control siRNA or Asef siRNA. Two days later, knockdown efficiency was examined by immunoblotting. Signal intensities of each band were quantified and are shown below each band as a relative strength to the control (in %) in B and C. (D-F) A431 cells were transfected with an empty pSuper vector (control) or pSuper-Asef. After puromycin selection, cells were further transfected with pRaichu-Rac1. After 3-6 hours of serum starvation, cells were stimulated with 25 ng/ml EGF. (D) Representative FRET images at the indicated time points are shown in the intensity-modulated display mode. (E) Normalized FRET:CFP ratios as described in the text. (F) From the peak values of the normalized ratio, the effect of Asef knockdown on EGF-induced Rac1 activation was calculated as described in the legend to Fig. 1D. The error bars indicate + s.d. Numbers of cells under each condition were as follows: control, n=13; Asef KD, n=14. ^*P<0.05, significant difference as compared with control (Student's t-test). (G) Control and Asef knockdown cells were starved for 6-12 hours, stimulated with 25 ng/ml EGF for 2 minutes or left unstimulated, and were examined for active Rac1 by pull-down assay. Experiments were repeated at least three times, and average values of the fold increase over control cells are given + s.d. ^*P<0.05, significant difference as compared with the contro (Student's t-test).