Sc 47724 hrp

Cell lysates were prepared in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate, pH 8.0) containing protease inhibitor cocktails (P8340, Sigma-Aldrich, St. Louis, MO). Cell lysates were boiled in SDS loading buffer, size fractionated by SDS-PAGE, and transferred onto a PVDF membrane (BioRad). The membranes were blocked for 1 hour at room temperature in TBST buffer containing 5% non-fat milk, then incubated overnight with the following primary antibodies: anti-AR mouse monoclonal (1:1000, sc-7305, Santa Cruz Biotechnology, Dallas, TX ), anti-PSA goat polyclonal (1:500, sc-7638, Santa Cruz Biotechnology), anti-PARP rabbit monoclonal (1:1000, # 9532S, Cell Signaling Technology, Danvers, MA), anti-cleaved caspase 3 rabbit monoclonal (1:1000, #9664S, Cell Signaling Technology) and anti-GAPDH mouse monoclonal (1:10000, sc-47724 HRP, Santa Cruz Biotechnology) antibodies. After three washes in TBST, membranes were incubated with secondary antibody (sc-2004, sc-2005, or sc-2354) diluted in blocking buffer for 1 hour at room temperature. Following three washes in TBST, the secondary antibody was detected by Enhanced Chemiluminescent (ECL) using an ECL kit (BioRad Laboratories, Hercules, CA) and signals on the membranes were visualized using a ChemiDoc Imaging System (Bio-Rad).