Fig. 6. The disruption of cell-cell adhesion promotes the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120^ctn. FRT cell monolayers kept overnight in serum-free medium were either left untreated (Ctrl), treated with 4 mM EGTA for 30 minutes (EGTA), pretreated with 10 µM PP2 for 1 hour before EGTA treatment (PP2+EGTA), or resuspended by treatment with 5 mM EDTA in PBS (Su), and then processed for immunoprecipitation and western blotting. (A) Rap1 immunoprecipitates (IP Rap1) were immunoblotted sequentially with antibodies against the indicated proteins (left panels). Equivalent loading of immunoprecipitated Rap1 was demonstrated by reprobing the blot for Rap1. In parallel, western blots of whole cell lysates were reprobed sequentially to assess total levels of the analyzed proteins (right panels). The multiple bands of p120^ctn represent distinct isoforms. Notice that immunoprecipitation of Rap1 from EGTA-treated cells resulted in the enhanced coprecipitation of a subset of E-cadherin-catenin complexes that did not contain p120^ctn. (B) Immunoprecipitations of Rap1 (IP Rap1) and E-cadherin (IP E-cadh) were immunoblotted sequentially with antibodies against the indicated proteins. Notice that PP2 blocked the increased formation of Rap1/E-cadherin-catenin complexes induced by EGTA treatment (left panels). The p120^ctn protein that coprecipitated with E-cadherin (p120^ctn, right panel) is the smaller but most abundant of the four p120^ctn isoforms expressed in FRT cells. #, E-cadherin full length; *, major E-cadherin cleavage products.