Figure 1. Effect of VP-16 and celastrol on AR in intact cells and cell-free extracts.

A: Effect of VP-16 on AR protein levels in LNCaP cells. Whole cell extracts prepared from LNCaP cells treated with 100 µmol/L VP-16 for indicated duration were subjected to Western blot analysis using AR (N20) antibody. β-actin was used as loading control. B: Effect of VP-16 on AR mRNA in LNCaP cells. AR mRNA expression during above treatment was determined by RT-PCR as described in Material and Methods. GAPDH was used as a control house keeping gene. C: AR proteolytic activity in cytosolic and nuclear fractions of celastrol-treated PC-3 cells. Cytosolic (Cyto) and nuclear (Nu) fractions (25 to 100 µg of protein) prepared from PC-3 cells treated with 5 µmole/L celastrol for 16 h were incubated with AR-containing nuclear fraction from LNCaP cells (LNAR) (25 µg of nuclear protein per incubation) for 3 h at 37 °C. AR protein remaining in individual incubation was then determined by Western blot analysis. Loading control β-actin levels showed an increase with increasing amount of PC-3 cytosolic and nuclear in the incubations 1 to 4 and 5 to 7, respectively. D: Effect of cytosolic and nuclear fractions from celastrol-treated PC-3 cells on purified recombinant human AR (rhAR). rhAR (2 ng) was incubated for 3 h at 37 °C with or without nuclear (Nu) or cytosolic (Cyto) fractions from celastrol-treated PC-3 cells. Residual rhAR in individual incubations was determined by Western blot analysis using AR (N20) antibody. E: Cytosolic fraction prepared from exponentially growing PC-3 cells treated with either the vehicle (non-Apop) or 5 µmol/L celastrol for 16 h (Apop) were incubated with rhAR (1 ng) for indicated duration and rhAR remaining in individual incubations was then determined by Western blot analysis using AR (N20) antibody. β-actin levels were used as loading control for cytosolic fractions.