(A) 293T cells were transfected with the expression vector encoding wildtype AR or Y267F AR or Y534F AR along with constitutively active Ack1 or empty vector. After 24 hours, protein extracts were immunoblotted with the antibody specific for phospho-Y267 AR or total AR. (B) COS7 cells were transfected with the vector encoding wildtype AR or Y267F AR or Y534F AR along with constitutively active Ack1 or activated Src or empty vector, as indicated. After 24 hours, protein extracts were immunoprecipitated with the AR antibody, then immunoblotted with the antibody specific for phospho-Y267 AR or phospho-Y534 AR or total AR, as indicated. (C) LNCaP cells were treated with EGF (100 ng/ml), heregulin (10 ng/ml), Gas6 (100 ng/ml), IL-6 (10 ng/ml), or bombesin (1 nM) for 60 min. Protein extracts were immunoblotted with the antibody specific for phospho-Y267 AR or total AR. (D) LNCaP cells were treated as above. Protein extracts were immunoprecipitated with the AR antibody, then immunoblotted with the antibody specific for phospho-Y534 AR. Blots shown are representative of three independent experiments.