Fig. 2. Estrogen-dependent c-Src activation is required for complex formation. (A) T47D cells grown to confluence and made quiescent as indicated (in Fig. 1), were left untreated or treated with 10 nM E2 for the indicated times (in seconds and minutes) and then detergent extracted. Cell extracts were immunoprecipitated with c-Src antibodies and immunoprecipitates subjected to kinase assay. Immunoprecipitates were run on 6% SDS-PAGE gel, dried and exposed for 3 hours at –80°C. Half of the immunoprecipitates was run in a parallel gel, transferred and blotted with antibodies to c-Src. (B) T47D cells in the same conditions as in A were treated with 10 nM estrogen in the presence of 5 µM PP1 for the indicated minutes. Cell lysates were immunoprecipitated with antibodies to estrogen receptor . Material co-immunoprecipitated with estrogen receptor were run on an 8% SDS-PAGE gel and immunoblotted with antibodies to p130Cas, c-Src and estrogen receptor . The data reported here are of a representative experiment out of three separate experiments. (C) T47D were treated for 3 minutes with 10 nM estrogen and cell extracts were immunoprecipitated either with antibodies to estrogen receptor or first with antibodies to Src and then to estrogen receptor . The immunoprecipitates were processed as described in B.