ARHGAP25 regulates the GTPase activity of Rac. (A) The investigated fragments of ARHGAP25. (B-D) Effect of full-length or truncated forms of ARHGAP25 on in vitro [γ-³²P]GTP-hydrolysis of Rac (B), Rho (C), and Cdc42 (D). Mean ± SEM of 4 separate experiments is shown (*P < .05 vs control). (E-L) Inhibition of EGF-induced ruffling by ARHGAP25 and p50RhoGAP. COS7 cells were transfected with control vector (E,I), cyan fluorescent protein (CFP)–tagged ARHGAP25R192A (F,J), CFP-ARHGAP25 (G,K), or CFP-p50RhoGAP (H,L). ARHGAP25, ARHGAP25R192A, p50RhoGAP, and CFP from control vector are shown in blue. Actin was stained with Alexa-568–labeled phalloidin (red). Stimulation was carried out with 0.1 μg/mL EGF for 20 minutes (I-L). EGF-induced ruffling is indicated by arrows. (M) Quantification of EGF-induced ruffling. Percentage of ruffled cells from 100 transfected cells was calculated. Mean + SEM of 4 (control vector, ARHGAP25R192A, and ARHGAP25) or 3 (p50RhoGAP) separate experiments are shown (*P < .05 compared with control).