Western blot analysis for NF-κB p65, Nrf-2 and HO-1

Protein (NF-κB, Nrf2, and HO-1) levels were analyzed by western blotting technique. For western blotting; epididymis, prostate, testes and vas deferens tissues of the rats were removed after sacrification to analyze the target protein expressions among the groups. Briefly, accurately weighed each tissue sample was homogenized in 1:10 (w/v) in 10 mM Tris-HCl buffer at pH 7.4, containing 0.1 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, and 5 μM soybean (soluble powder; Sigma, St. Louis, MO, USA) as trypsin inhibitor. Tissue homogenate was centrifuged at 15,000 g at 4 °C for 30 min, and the supernatant was transferred into fresh tubes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer containing 2 % β-mercaptoethanol was added to the supernatant. Equal amounts of protein (20 μg) were electrophoresed and subsequently transferred to nitrocellulose membrane (Schleicher and Schuell Inc., Keene, NH, USA). Nitrocellulose blots were washed twice for 5 min in phosphate buffered saline (PBS) and blocked with 1 % bovine serum albumin in PBS for 1 h prior to the application of primary antibody. Rat antibodies against NF-κB 65, Nrf-2 and HO-1 were purchased from Abcam (Cambridge, UK). Primary antibody was diluted (1:1000) in the same buffer containing 0.05 % Tween-20. The nitrocellulose membrane was incubated overnight at 4 °C with protein antibody. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK). Specific binding was detected using diaminobenzidine and hydrogen peroxide as substrates. Protein loading was controlled using a monoclonal mouse antibody against β-actin antibody (A5316; Sigma). Band intensities of the proteins were quantified by densitometric analysis using an image analysis system (Image J; National Institute of Health, Bethesda, USA). Samples were analyzed in quadruplicate, and a representative blot is shown in the respective figures. Results were normalized to the β-actin expression in each group as percent of control.