Fig. 1. Paxillin is required for PTP-PEST inhibition of cell spreading. (A) Paxillin^+/+ and paxillin^–/– cells were transfected with PTP-PEST and GFP or with GFP control vector alone. Cells were harvested and maintained in suspension for 60 minutes at 37°C and then replated on fibronectin-coated slips for 45 minutes or overnight. Cells were fixed at 45 minutes and stained with rhodamine phalloidin. The number of unspread transfected cells (GFP-positive) was evaluated (see Materials and Methods for details). Expression of PTP-PEST inhibited spreading of the paxillin^+/+ but not paxillin^–/– cells. Cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar, 25 µm. (B) Quantification of PTP-PEST-dependent inhibition of spreading was measured as the percentage of round GFP-positive cells. A minimum of 100 cells were counted per condition in three independent experiments. Results show mean±s.d. **P<0.001. (C) Western immunoblotting of total cell lysates demonstrates the absence of paxillin and upregulation of Hic-5 in paxillin^–/– cells compared with the paxillin^+/+ control cells.