Fig. 2. A direct paxillin–PTP-PEST interaction is necessary for PTP-PEST to regulate cell spreading. (A) Paxillin^–/– cells were transfected with PTP-PEST and paxillin, PTP-PEST and the paxillin C523S mutant (which is defective for PTP-PEST binding) or the PTP-PEST Pro II mutant (defective for paxillin binding) alone, in the absence or presence of paxillin. Cells were plated on fibronectin and assayed for spreading at 45 minutes post plating. Co-transfection with GFP allowed identification of transfected cells. Cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar, 25 µm. Perturbation of the paxillin-PTP-PEST interaction blocks PTP-PEST inhibition of cell spreading. (B) Quantification of round, GFP-positive cells observed in A. Results show the mean±s.d. A minimum of 100 cells were counted in three independent experiments. **P< 0.001. (C) Western immunoblot analysis confirms equivalent expression of paxillin and PTP-PEST constructs. (D) Localization of paxillin and paxillin C523S mutant to focal adhesions in paxillin^–/– cells was visualized by indirect immunofluorescence microscopy. Cells were also stained with antibody against vinculin to visualize focal adhesions. Bar, 15 µm.