Fig. 3. p130Cas over-expression modulates estrogen-dependent c-Src activation. T47D cells were transiently transfected with empty vector or the p130Cas cDNA by the Lipofectamine 2000 method. 24 hours post-transfection, cells grown to confluence and made quiescent (as indicated in Fig. 1) for an additional 24 hours, were left untreated or treated with 10 nM E2 for the indicated times and then detergent extracted. Cell extracts were immunoprecipitated with c-Src antibodies and immunoprecipitates subjected to kinase assay. Immunoprecipitates were run on a 6% SDS-PAGE gel, dried and exposed at –80°C. Half of the immunoprecipitates was run in a parallel gel, transferred and blotted to antibodies with c-Src and re-blotted with polyclonal antibodies to myc epitope (lower panel). The relative amount of c-Src autophosphorylation was determined by densitometric analysis (on the right) of the autoradiographs. In each case, the autophosphorylation signal was normalized for the corresponding amounts of c-Src immunoprecipitated and expressed as arbitrary units relative to the untransfected and untreated control. The statistical significance of the different values was calculated using Student's t-test (^*) (P<0.05). Similar results were obtained in two other experiments.