Fig. 3. PKC^-/- fibroblasts show reduced recruitment of focal adhesion components on fibronectin. (A) Compartmentalization of focal adhesion components in PKC^+/+ (WT) and PKC^-/- fibroblasts. Cells were cultured until 80% confluence on fibronectin. Fibronectin is a major component of the provisional extracellular matrix deposited during wound healing (Grinnell, 1984). Triton-soluble and -insoluble protein extracts were prepared and subjected to SDS/PAGE as described in the Materials and Methods. Recruitment of FAK and β-actin is not affected. Western blot analyses (25 µg/lane) were then performed with anti--SMA, anti-β-actin, anti phospho-FAK, anti-FAK, anti-Rac1, anti-paxillin and anti-vinculin antibodies. There was reduced recruitment of Rac, paxillin, vinculin, phospho-FAK and -SMA to the insoluble (cytoskeleton) fraction of the cell. Recruitment of FAK and β-actin was not affected. FAK phosphorylation was not completely impaired in the absence of PKC. Densitometry was performed (n=3, average±s.d. is shown, ^*P<0.05 relative to wild-type control). (B) Interaction between paxillin and FAK in PKC^+/+ and PKC^-/- fibroblasts. Cell extracts (25 µg) from cells cultured in an analogous fashion to those in A were immunoprecipitated with -FAK or -paxillin (-PAX) or normal goat serum control (C), as indicated. The resultant protein extracts were subjected to SDS/PAGE, and immunoprecipitated FAK was detected with a -FAK antibody by western analysis. FAK shows reduced interaction with paxillin in the absence of PKC.