Validation of the RPCI-5αR3 antibody. A: 5α-reductase-3 mRNA expression level in LNCaP, C4-2, LAPC-4, and CWR-R1 cells. 5α-reductase-3 mRNA expression levels in the CaP cell lines were normalized to the level of expression of 5α-reductase-3 in the PWR-1E benign prostate epithelial cell line (discontinuous red line). B: PWR-1E, LNCaP, C4-2, LAPC-4, CWR-R1, and CHO-K1 protein lysates (50 μg) were immunoblotted and 5α-reductase-3 protein expression was detected using the RPCI-5αR3 antibody. C: CWR-R1 lysates (50 μg) were immunoblotted and 5α-reductase-3 protein expression was analyzed using RPCI-5αR3 antibody at a concentration of 1 μg/ml or SRD5A3 (Sigma) antibody at concentrations 1 μg/ml and 5 μg/ml. D: Immunostaining analyses were performed in CWR-R1 using the RPCI-5αR3 antibody. Preincubation of the RPCI-5αR3 antibody with the inhibitor peptide (1 and 10 μg/ml) confirmed specificity. Absence of primary antibody provided negative control (Control).