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Fig. 1. Effect of Sos, Vav2, and Tiam1 depletion on EGF-induced Rac1 activation. (A) Schematic, showing the current view of regulation of Rac1 by EGF. (B) A431 cells were transfected with either Sos1- and Sos2-specific siRNAs, Vav2-specific siRNA or Tiam1-specific siRNA. Forty-eight hours after transfection, cells were lysed and subjected to immunoblot analysis using antibodies against Sos1, Sos2, Vav2 and Tiam1. (C) A431 cells were transfected with siRNA as indicated. One day after siRNA transfection, cells were further transfected with pRaichu-Rac1. After serum starvation for 6 hours, cells were stimulated with 25 ng/ml EGF and examined for Rac1 activity by FRET microscopy as described in the text. Averages of the fold increase after EGF addition over the control cells are given + s.d. Numbers of cell examined under each condition were as following: control, n=85; Sos1,2 KD, n=13; Vav2 KD, n=9; Tiam1 KD, n=90. *P<0.05, significant difference as compared with the control (Student's t-test). (D) Control and knockdown cells were starved for 6-12 hours, stimulated with 25 ng/ml EGF for 2 minutes or left unstimulated, and examined for active Rac1 by pull-down assay. Experiments were repeated at least three times, and average values of fold increase over the control cells are given + s.d. *P<0.05, significant difference as compared with the control (Student's t-test).
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