Effect of progestins on Src and Erk activity in COS-7 cells transfected with PR (wild type and mutants) and ERα expression vectors. (A) COS-7 cells were transfected with pSG5 control plasmid (lanes 1 and 2), wild-type PRB (lanes 3 and 4), ERα (lanes 5 and 6), or both PRB and ERα (lanes 7 and 8) expression vectors and treated for 5 min with 10 nM R5020 or with ethanol. Cell lysates were immunoprecipitated with anti-Src antibodies. The immunoprecipitates were assayed for Src activity with acidified enolase (en) as a substrate (upper panel). The expression of PRB and ERα was verified by immunoblotting of total cell lysates with anti-PR (middle panel) or anti-ERα (lower panel) antibodies. (B) COS-7 cells were transfected with PRB (wild type or mutant PRmPro or PRΔPro) expression vectors alone or together with ERα cDNA. Cells were treated for 5 min with 10 nM R5020 or with ethanol. Lysates were prepared, divided into two aliquots, and immunoprecipitated with anti-Src or anti-Erk antibodies. The immunoprecipitates were assayed for Src and Erk activity with acidified enolase (en) or MBP as substrates, respectively. (C) Expression of PRB (wild type and mutants) and ERα was verified by immunoblotting of total cell lysates with anti-PR (upper panel) or anti-ERα (lower panel) antibodies.