Fig. 4. Paxillin N-terminus, LD4 motif and tyrosine phosphorylation sites Y31 and Y118 are required for PTP-PEST-dependent inhibition of spreading. Paxillin^–/– cells were transiently transfected with GFP, paxillin alone or together with PTP-PEST, or the following paxillin constructs with and without PTP-PEST: (A) LIM1-4, (B) LD4, (C) LD1 or LD2 and (D) Y31/118F, and plated on fibronectin-coated coverslips. Cells were fixed after 45 minutes and processed for indirect immunofluorescence. Results represent the percentage of round GFP-positive cells. Results show the mean±s.d. A minimum of 100 cells were counted in three independent experiments. **P<0.001, * P<0.05 (E) GFP-transfected paxillin^–/– cells that had also been transfected with PTP-PEST, paxillin and PTP-PEST, PTP-PEST and Pax C523S, PTP-PEST and Pax LD4, PTP-PEST and Pax Y31/118F, or PTP-PEST and Pax Y31/118F LD4 were plated on fibronectin-coated coverslips for 45 and 120 minutes. Cells were fixed and processed for indirect immunofluorescence as described above. Total cell area was obtained from these images using Spot^TM RT software. Results represent the mean±s.d. of a minimum of 50 cells for each time point and three independent experiments. **P<0.001.