6. Western blot analysis

Western blot analyses were used to determine the levels of AR protein in testis and prostate tissues. Tissues frozen in liquid nitrogen were homogenized in lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 2 mM EDTA, 1 mM EGTA, and 1% Triton X-100). The protein concentration was determined using the bicinchoninic acid protein assay (Pierce Chemical Co., Dallas, TX, USA). Forty micrograms of total cellular protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. After blocking for 1 hour at room temperature with 5% skim milk in 0.1% tris-buffered saline with Tween 20, the membrane was incubated with β-actin antibody (1:1,000 dilution; Assay Designs, Ann Arbor, MI, USA) and anti-AR antibody (AR 1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4℃. The β-actin was used as an internal control. Detection of bound antibody on each blot was evaluated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G) visualized by hybond enhanced chemiluminescence (Amersham, Arlington Height, IL, USA). The densitometric analysis of band intensity was based on the Luminescent Image Analysis System (LAS-3000; Fujifilm, Tokyo, Japan) and analyzed using Multi Gauge 3.0 software (Fujifilm).