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Figure 3


Fig. 3. G{alpha}12/Src activation disrupts interactions of claudin 1 and occludin with ZO-1. (A) Specificity of ZO-1 immunoprecipitations. G{alpha}12-MDCK cells were cultured +/–dox +/–PP2 and immunoprecipitated with rat monoclonal R46 antibody or the equivalent amount of ascites control. Western blot for ZO-1 in cell lysates and immunoprecipitations using the identical antibody is shown. (B) G{alpha}12- and QL{alpha}-12 MDCK cells +/–dox were immunoprecipitated with ZO-1 antibody as described in the Materials and Methods. Immunoprecipitates were analyzed by western blotting to ZO-1 followed by reprobing with antibodies to other TJ proteins (ZO-2, occludin and claudin 1). A parallel analysis was performed in QL{alpha}12-MDCK cells pre-treated with the Src inhibitor PP2 (10 µM) added at the time of switch to –dox medium. (C) Bar graphs summarizing the fraction of TJ protein remaining associated with ZO-1. The results are the mean ± s.e.m. of three experiments normalized to the ZO-1 immunoprecipitates (+dox). *P<0.05.