FIGURE 2.

Calpain expression and activity in prostate-derived cells. A, inhibition of calpain activity in R1 cells with calpeptin (40 um) for 48 h decreases the expression of the LMW-AR (relative to FL-AR) by 55% in the absence of androgen (Ad) and 43% in the presence of androgen. B, top panel, Western blot analysis of calpain 2 levels in nontransformed and tumor prostate cells. Bottom panel, Western blot analysis of calpastatin levels in nontransformed and tumor cells. GAPDH served as a loading control. C, calpain-dependent proteolysis of FAK from a 120-kDa to a 90-kDa form and ultimately smaller forms is indicative of calpain activity. FAK proteolysis is greater in R1 than in Rv1 cells and is greater in both cells in the absence of androgens. D, calpain 2 siRNA down-regulated calpain 2 protein levels 144 h post-transfection in R1 cells. The down-regulation of calpain 2 expression by calpain 2 siRNA reduced the LMW-AR (relative to FL-AR) by 54% in the absence of androgen and 39% in the presence of androgen. Calpain-dependent proteolysis of FAK was also decreased. E, expression of CLDN4 in R1 cells culture in androgen-depleted media, following a 2-h stimulation with DHT and a 24-h treatment with 60 μm calpeptin was assessed by real-time PCR. CLDN4 expression was standardized to GAPDH. Error bars represent S.D. p < 0.05. Ab, antibody; siC, control siRNA; siCalapin, calpain 2 siRNA.