Fig. 8. Asef phosphorylation at Tyr94 has an essential role in EGF-induced activation of Rac1 and Cdc42. (A) A431 cells were transfected with vectors expressing wild-type Asef (WT) and siRNA-resistant Asef (WT^R) were harvested at 48 hours after transfection and analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. pSuper-Asef is an shRNA expression vector targeting Asef; pERedNLS-3HA-Asef-WT is a wild-type Asef expression vector; and pERedNLS-3HA-Asef-WT^R is a wild-type Asef expression vector carrying RNAi-resistant nuleotide substitutions. ^R, siRNA-resistant expression plasmids. (B) A431 cells were transfected with expression vectors as indicated below each bar (WT^R, pERedNLS-3HA-Asef-WT^R; Y94F^R, pERedNLS-3HA-Asef-Y94F^R). After selection with puromycin, cells were transfected with pRaichu-Rac1 for 24 hours. After 3-6 hours of serum starvation, cells were stimulated with 25 ng/ml EGF and analyzed as described for Figs 2 and 3. Numbers of cells examined for each condition were as follows: control, n=22; Asef KD, n=21; Asef KD and Asef WT^R; n=11; Asef KD and Asef Y94F^R, n=11. ^*P<0.05, significant difference over the control cells (Student's t-test). ^*P<0.05. (C) Experiments were performed as in B except that pRaichu-Cdc42 was used instead of pRaichu-Rac1. Numbers of cells examined for each condition were as follows: control, n=10; Asef KD, n=10; Asef KD and Asef WT^R, n=14; Asef KD and Asef Y94F^R, n=11.