Western blot analysis for detection of phosphorylated ASK1 and p38 MAPK

Liver segment was suspended in homogenization buffer (25 mM Tris-HCl, pH = 7.0, 10% Triton-X100) containing protease inhibitors and phosphatase inhibitors (protease and phosphatase inhibitor cocktail, Calbiochem), and homogenized using a tissue homogenizer (Yellow line DI 18 basic, IKA, Germany). The liver tissue homogenates were centrifuged at 10,000 rpm for 15 min. The supernatant was collected and used to prepare protein for Western blot analysis. The amount of protein was determined by the Bio-Rad protein assay method. Expression of ASK1 and p38 MAPK was analyzed by Western blot. Thirty micogram protein of each sample was heated at 100℃ for 5 min with a loading buffer containing 0.125 M Tris–HCl (pH 6.8), 20% glycerol, 4% SDS, 10% mercaptoethanol, and 0.002% bromophenol blue. It was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gel and the Bio-Rad minigel system. The proteins were electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Blotting membranes were incubated with 3% bovine serum albumin in tris-buffered saline with tween (TBST) (10 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-20) and probed with corresponding primary antibodies of anti-ASK1 and anti-p38 MAPK (CST, Beverly, MA, USA) at 4℃ overnight at a dilution 1:500 or 1:1000. Blotting membranes were washed three times with phosphate buffered saline and then incubated with horseradish peroxidase-coupled secondary anti IgG monoclonal antibody (Santa Cruz biotechnology, USA) for 2 h at room temperature. Membranes were exposed to films, and the bands were visualized using an enhanced chemiluminescence reagent (Amersham™, UK). These bands were quantitated by densitometry (UVP Upland, CA, USA). In all Western blot experiments, β-actin was used as an internal control for equal protein loading. Western blots were repeated three times for each protein.