Figure 5.

Kinetic analysis of dl 853–963 and dl 965–1012 phosphorylation upon cell adhesion. (A) Cells expressing dl 853–963 were held in suspension for 45 min in serum-free medium and then plated onto fibronectin- or poly-l-lysine–coated dishes. Cells in culture (lane 1), cells in suspension (lane 2), and cells plated onto fibronectin for 1 h (lane 3), 2 h (lane 4), or 3 h (lane 5) or cells plated onto poly-l-lysine for 3 h (lane 6) were lysed. FAK was immunoprecipitated using BC4, and the immune complexes were analyzed by Western blotting for phosphotyrosine (top). Endogenous wild-type FAK and mutant FAK are indicated by arrowheads. The blot was stripped and reprobed with BC4 (bottom). (B) Cells expressing dl 965–1012 were held in suspension for 4 h in serum-free medium and then plated onto fibronectin- or poly-l-lysine–coated dishes. Cells in culture (lane 1), cells in suspension (lane 2), and cells plated onto fibronectin for 1.5 h (lane 3) or 3 h (lane 4) or cells plated onto poly-l-lysine for 3 h (lane 6) were lysed. To test for the effects of serum, cells were adhered to fibronectin in serum-free medium for 1.5 h and then stimulated with culture medium for 1.5 h before lysis (lane 5). FAK was immunoprecipitated using BC4, and the immune complexes were analyzed by Western blotting for phosphotyrosine (top). The blot was stripped and reprobed with BC4 (bottom).