(A) LNCaP, CWR22Rv1, LAPC4 or VCaP cells were treated with 0, 1, or 10 nM DHT for 24h and AR or β-actin were immunoblotted. (B) VCaP cells were treated with/out DHT for 4h, 8h, or 24h and AR, PSA, or β-actin were immunoblotted. (C) VCaP cells were treated with 0, 0.1, 1, or 10 nM DHT and with 0, 10, or 40 μM bicalutamide for 24h and immunobloted for AR, Ser 81 phosphorylated AR, PSA, or β-actin. (D) VCaP or LNCaP cells were pre-treated with/out 10 nM DHT for 24h and then treated with MG115/MG132 for 4h. (E) VCaP or LNCaP cells were pre-treated with/out DHT for 2h and then treated with cycloheximide (10 ng/mL) for 0, 2, 4, or 6h. (F) VCaP or LNCaP cells were transiently transfected with empty vector or 3×Flag-AR. After 24h, cells were treated with/out 10 nM DHT for 24h (note: the prostate cancer cells were steroid-depleted by culturing in medium with charcoal/dextran stripped serum, CSS, for 3d before treatments in all experiments). See also Figure S1.