FIGURE 1.

Ser-85 phosphorylation of paxillin is dependent on cell adhesion. HeLa cells without (A) or with transfection with HA₃-paxillin (paxillin or S85A) for 36 h (C, D, and E) were collected, rolled over at 37 °C for 1 h, and maintained in suspension (Sus) for 2 h. Cells were reseeded onto culture dishes newly precoated with collagen I (10 μg/ml) for the indicated times prior to harvesting and immunoblot analysis of whole cell lysates with the indicated antibodies (A–C) or reseeded on collagen I-precoated coverslips for 15 or 30 min before processing for indirect immunostaining or actin staining (D and E). Anti-HA tag antibody (green; D) or anti-HA tag (green) and anti-phospho-Tyr-397 FAK (pY³⁹⁷FAK; red) primary antibodies and the appropriate secondary antibodies (E) were used. Scale bars = 20 μm (D and E). B, HeLa cells transfected with HA₃-paxillin (WT or S85A) for 36 h were kept in suspension or reseeded onto culture dishes precoated with 10 μg/ml fibronectin (FN), collagen I (CL), or laminin I (LN) for 30 min as described above. The cells were then harvested, and whole cell lysates were subjected to immunoblot analysis for the indicated molecules. The data represent three independent experiments. pS⁸⁵Pax, phospho-Ser-85 paxillin; Con, control.