Phosphorylation of stromal cell FAK and paxillin are increased after contact with RAMOS-wt or RAMOS-4c. Stromal cells were incubated in medium alone (−) or in contact with RAMOS-wt (wt) or RAMOS-4c (4c) for 5 minutes at 37°C. Stromal cells and RAMOS cells were collected, lysed, and immunoprecipitated and immunoblotted as detailed in the Materials and Methods. (A) Immunoprecipitation of 1 mg of cell protein/sample with rabbit preimmune IgG (cont) or with h-FAK1 polyclonal antibody (FAK). Preimmune IgG immunoprecipitation was performed on pooled stromal cell lysate. RAMOS indicates FAK immunoprecipitate from pooled lysate of RAMOS-wt and RAMOS-4c that had been washed from stromal cells. (Upper panel) Immunoblotting with antiphosphotyrosine. (Lower panel) Immunoblotting with rabbit anti-FAK C-903. (B) Densitometric comparison of FAK phosphorylation in stromal cells after contact with RAMOS-wt (wt) or RAMOS-4c (4c) normalized to FAK phosphorylation of stromal cells incubated in medium (−) in four separate experiments. Error bars represent the standard error. (C) Immunoprecipitation of 0.6 mg cell protein/sample with antipaxillin. RAMOS indicates paxillin immunoprecipitate from pooled lysate of RAMOS-wt and RAMOS-4c that had been washed from stromal cells. (Upper panel) Immunoblotting with antiphosphotyrosine. (Lower panel) Immunoblotting with antipaxillin. (D) Densitometric comparison of paxillin phosphorylation in three separate experiments (as for FAK in [B]).