Anti ho 1 sc 10789

The epididymal tissues (~100 mg) were homogenized in 300 µL of ice-cold protein lysis buffer [50 mM Hepes-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 2.5 mM EGTA (pH 8.0), 1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM Na₃VO₄, 1 mM DTT, 1% NP-40, 10% glycerol, 0.2 mM PMSF and Protease inhibitor cocktail (Sigma, USA)] using the Tissue Lyser system (Qiagen, USA) with 5 mm sterile stainless steel beads. After centrifugation for 30 min at 10,000 ×g at 4℃, the protein content of the supernatant was measured using a protein assay kit (Bio-Rad, USA). Equal amounts of protein were loaded into the lanes of an SDS-PAGE gel, separated and then transferred to a PVDF membrane using a semi-dry electrotransferring unit (Bio-Rad). After blocking with 5% nonfat milk or bovine serum albumin in TTBS, membranes were probed with specific antibodies diluted in TTBS with 5% nonfat milk or bovine serum albumin as follows: anti-catalase (ab-16731, Abcam, USA), anti-HO-1 (sc-10789, Santa Cruz Biotechnology, USA), anti-p-JNK (s-9251, Cell signaling, USA), anti-70-kDa heat shock cognate protein (HSC70; Santa Cruz Biotechnology) or anti-beta-actin (A5441, Sigma, USA). The membranes were then incubated with an IgG-peroxidase-conjugated secondary antibody for chemiluminescent detection. The band intensities were quantified using Quantity One software (Bio-Rad).