Fig. 5. E-cadherin adhesive function modulates Src activity, which in turn is required for Rap1 activation in response to the disruption of AJs. (A,B) FRT cell monolayers kept overnight in serum-free medium were either subjected to the Ca²⁺ switch procedure [(Ctrl), (EGTA), (EGTA+Ca²⁺)], resuspended by 5 mM EDTA treatment in PBS (Su), or pretreated for 1 hour at 37°C with the specific SFK inhibitor PP2 (10 µM) before EGTA treatment (PP2+EGTA). Cell extracts were then prepared, and Src was immunoprecipitated from lysate supernatants with a c-Src-specific mAb. Src immunoprecipitates were then washed and divided equally; one half was subjected to an in vitro autophosphorylation assay (A), and the other half was used for a quantitative western blotting assay to confirm equal loading of immunoprecipitated Src proteins (B). Notice that the Ca²⁺ switch procedure clearly affected the kinase activity of endogenous Src. The effectiveness of cell resuspension in activating, and of PP2 treatment in inhibiting, Src activity is also shown. (C) FRT cells were either left untreated (Ctrl), treated with 4 mM EGTA for 30 minutes (EGTA), or pretreated with PP2 (10 µM) for 1 hour before EGTA treatment (PP2+EGTA). Active Rap1 levels were then measured by the pull-down assay. Notice that SFK inhibition by PP2 completely blocked the activation of Rap1 induced by EGTA treatment.