Fig. 7. PTP-PEST regulates Rac1 activation through an interaction with paxillin. (A) PTP-PEST^–/– cells or (B) paxillin^–/– cells were co-transfected with Myc-tagged wild-type Rac1 together with GFP, PTP-PEST, PTP-PEST and paxillin, or PTP-PEST and paxillin C523S, and either held in suspension or plated on fibronectin-coated culture dishes for 60 minutes. The level of activated Rac was determined using a GST-PAK-PBD pull-down assay as described in Materials and Methods. Total and active Rac levels were visualized by western blotting and quantitative densitometry was performed. (C,D) Results show the mean±s.d. of three independent experiments. PTP-PEST inhibited adhesion-induced Rac activation only in the presence of paxillin. This effect was reversed by introduction of the paxillin C523S mutant defective in PTP-PEST binding. **P<0.001.