Fig. 5. Binding of Cdc42, Tc10 and RhoT to the CRIB motif of N-WASP leading to the Arp2/3-complex-mediated actin polymerization. (A) Yeast two-hybrid interaction assay. The yeast strain Y190 was transformed with wt, constitutively active or dominant-negative mutants of the small GTPases in bait pGBT9 vector and with the N-terminal portion (amino acids 1-275) of N-WASP containing the CRIB motif in prey pACT2 vector. The interaction was analyzed by ß-galactosidase colony-lift filter assay. (B) Pull-down assay. GST-tagged small GTPases loaded with GTPS or GDP were immobilized to glutathione-Sepharose, and then the lysate from Balb/3T3 cells transfected with HA-tagged N-WASP was applied to the resin. Proteins bound to the small GTPases were eluted, and N-WASP was detected by immunoblotting (a). Used GST-small GTPases were analyzed by SDS-PAGE (b). (C) Fluorometric actin polymerization assay. Protein mixtures contain 60 nM Arp2/3 complex, 100 nM His-N-WASP, 200 nM GST-VCA or 400 nM small GTPases in X buffer. The reaction was started by adding the mixture of 2 µM unlabeled actin and 0.2 µM pyrene-actin to the preincubated protein mixtures, and fluorescence change was measured at 407 nm.