Amersharm

The liver tissue and cell lysates were homogenized in mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO. USA) in the presence of protease inhibitor cocktail (Sigma) and phenyl methane sulfonyl fluoride (Sigma, PMSF). The lysates were centrifuged at 12,000 rpm at 4 °C for 20 min. The protein contents of the supernatants were determined using protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA) based on the manufacturer’s instructions. The same concentration of protein was separated electrophoretically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Amersharm, GE Healthcare Life Science, Germany). The antibodies used for western blotting included anti-SREBP-1 (1:1000; Abcam, Cambridge, UK), anti-ACC (1:1000; Cell Signaling Technology), anti-FAS (1:1000; Cell Signaling Technology), anti-AMPKα (1:1000; Cell Signaling Technology), anti-phospho-AMPKα (Thr172) (1:1000; Cell Signaling Technology), and anti-β-actin (1:2500; Abcam). The protein bands were visualized following an enhanced chemiluminescence method using an ELC kit (Millipore, USA). The bands were quantified using Quantity 1 version 4.6.7 software (Bio-Rad Laboratories).

Proteins were extracted with the mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO, USA) containing the protease inhibitor cocktail (Sigma) and phenyl methane sulfonyl fluoride (PMSF, Sigma). Protein concentrations in supernatants collected after centrifugation of protein lysates were established using protein assay dye reagent concentrates (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were split by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersharm, GE Healthcare Life science, Germany). Primary antibodies were reacted with anti-occludin (1:1000; Invitrogen, USA), anti-claudin-1 (1:1000; Invitrogen), anti-ZO-1 (1:1000; Invitrogen), iNOS (1:500; Santa cruz, USA), COX2 (1:1000; Cell signalling), 4-hydroxynonenal (4HNE) (1:1000; Abcam, UK), AMPK (1:1000; Cell signalling), phospho-AMPK (1:1000; Cell signalling), and anti-β-actin (1:2500; Abcam, Cambridge, UK) at 4 °C overnight, respectively. After secondary antibody reaction for 2 h at room temperature, target protein bands were visualized by an enhanced chemiluminescence method (ECL, Millipore, Boston, MA, USA). Quantity of luminescence was performed using Quantity 1 version 4.6.7 software (Bio-Rad Laboratories, Hercules, CA, USA).