Hybond enhanced chemiluminescence

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).

B-NHL cell lines were incubated with or without 100 pM of anti-CD20-hIFNα or rituximab at 37°C for 18 h. Western blot analysis was performed as previously described (21 (link)). Briefly, cell extracts for protein analysis were prepared by lysing 2×10⁶ cells on ice with cold 200 μl of radioimmunoprecipitation assay buffer [1% NP40, 0.1% SDS, 0.5% deoxycholic acid, complete protease inhibitor cocktail tablets (Roche Diagnostic Co.), and 1X PBS]. Lysates were transferred to microcentrifuge tubes and sonicated in the Sonicator, model W-220F (Heat-System Ultrasonic, Inc.) for 10 sec. Lysates were centrifuged at 12,000 × g at 4°C for 5 min. Protein concentration was quantified using the Bio-Rad protein assay (Bio-Rad Laboratories). Gel loading buffer Bio-Rad (Bio-Rad Laboratories) was added to the cell lysates at a 1:1 volume. Samples were boiled for 5 min and were separated on 12% SDS-polyacrylamide mini-gels and transferred to a nitrocellulose membrane Hybond enhanced chemiluminescence (Amersham Pharmacia Biotech) in Trans-Blot SD semidry Transfer Cell System (Bio-Rad).