LNCaP C-33 cells were plated in the regular culture medium for 72 h. After steroid starvation for 48 h, cells were treated with or without DHT (10 nM), 5 µg/ml actinomycin D (Act D) or 10 µg/ml cyclohexamide (CHX) as specified in the figure for 24 h. After harvesting, cell lysates were analyzed by western blotting with Abs against total Shc, prostate-specific antigen (PSA), androgen receptor (AR) and β-actin protein, respectively. The level of β-actin protein was detected as a loading control. The intensity of p66Shc hybridization band was semiquantified, and the ratio to the corresponding β-actin protein was calculated and then normalized to that of control LNCaP cells which received the solvent alone. The figure is a representative of four sets of independent experiments. SD, Standard deviation.