Fig. 6.

Direct interaction between PLC-γ1 and Rac1, not the other factors, is critical for EGF-induced Rac1 activation. A, Binding affinity of wt Rac1 and mutants Rac1 PP/AA, N17, and L61 to GTP-γS. COS-7 cells were transfected with GFP-tagged wt Rac1 or the mutants. The cell lysates were treated with GTP-γS for 15 min and were then pulled down by GST-PAK. Active GTP-Rac1 is visualized by immunoblotting with GFP antibody. B, Inhibition of PLC-γ1 phospholipase activity and the effects on EGF-induced Rac1 activation. Cos7 cells were transfected with PLC-γ1 and PLC-γ1ΔSH3. The cells were treated with U73122 and stimulated with EGF. The Rac1 activation was determined by GST-PAC pulldown. C, Quantification of the data from at least four independent experiments as described in panel B.