Western blot analysis

HepG2 cells were treated with 1 mmol/l FFAs alone or together with compound 2 (25 µmol/l) for 24 h. Cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing 1 mmol/l phenylmethane sulfonylfluoride for 20 min on ice. Subsequently, cell lysates were centrifuged at 12,000 × g for 10 min at 4°C. The protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (40 µg) of extracted protein samples were separated by 15% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk for 1 h at room temperature (~22°C), and then incubated with anti-GAPDH, anti-ERp57 and anti-FAS primary antibodies at 4°C overnight. Following washing three times with TBST (TBS containing 0.1% Tween-20; cat no. P0231; Beyotime Institute of Biotechnology), the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG secondary antibodies at room temperature for 2 h. Protein bands were visualized by enhanced chemiluminescence using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.).