Western Analysis

LNCaP cells were plated in RPMI supplemented with 8% CFS. Following 48 hr incubation, cells were treated with hormone for 18 hr. Cells were lysed (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.05% SDS, 5 mM EDTA, 50 mM NaF, 15 mM Na-pyrophosphate, 10 mM β-glycerophosphate, 2 mM Na-orthovanadate, 1X protease inhibitor cocktail) and cleared whole cell extracts were analyzed by Bradford assay. 50 μg of protein per sample were resolved by SDS-PAGE (8%), transferred to nitrocellulose membrane, and analyzed by western blot using antibodies to AR (N-20, Santa Cruz) and β-actin (Sigma) per manufacturer’s instructions.