Gsh px

The liver tissues were homogenized with lysis buffer containing a protease inhibitor cocktail. The homogenates were centrifuged at 12,000 rpm for 20 min at 4°C. After the supernatant was collected, the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Irvine, CA, USA). An equal amount of proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was incubated with 5% skim milk for 50 min at room temperature and then washed with PBS-T. The membrane was incubated with a primary antibody overnight at 4°C and then washed with PBS-T. The primary antibodies used were: β-actin (1:1,000; Cell Signaling, Beverly, MA, USA), NF-κB p65 (1:500, Abcam, Cambridge, UK), iNOS (1:500, Merk, Kenilworth, NJ, USA), COX-2 (1:1,000, Cell Signaling, Beverly, MA, USA), IL-1β (1:500; Bioss Antibodies, Boston, MA, USA), SOD (1:500; Santa Cruz, Dallas, TX, USA), catalase (1:500; Santa Cruz), and GSH-Px (1:500, Santa Cruz). The membranes were incubated with secondary antibodies for 1 h. The immuno-complexes were visualized by enhanced chemiluminescence solution (ELPIS Biotech, Daejeon, Korea) and bands were visualized with a chemiluminescent imaging system (Davinci Chemi, Seoul, Korea).