Goat anti rabbit horseradish peroxidase secondary antibody

For western blotting, the frozen liver tissues were homogenized in protein lysis buffer (PRO-PREPTM, Intron Biotechnology, Seongnam, Korea), and then centrifuged (13,000 g at 4°C for 30 min). After the supernatants of the liver lysates were collected, their respective protein contents were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The 50 μg of total protein were loaded on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature. The blocked membranes were then incubated overnight with diluted primary antibodies (diluted 1:1,000 with 5% BSA in TBS-T) at 4°C. Primary antibodies used include monocyte chemoattractant protein 1 (MCP-1) and anti-tumor necrosis factor alpha (TNF-α; Cell Signaling Technology, Danvers, MA, USA). After washing with 1× TBS-T, the membrane was incubated with goat anti-rabbit-horseradish peroxidase secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 3 h at 4°C. The bands were detected using enhanced chemiluminescence kit (GE Healthcare). Target protein expression was normalized to that of β-actin.