Western Blot Analysis of Liver Proteins

Whole and nuclear proteins were extracted from liver and HepG2 cells as described previously [10 (link)]. Protein concentration was quantified with the BCA method according to the supplier's instructions (Sigma). 50 μg of protein was added to 8% SDS-PAGE and electroblotted onto PVDF membranes (Pall Corporation, Pensacola). The membranes were blocked and incubated with specific antibodies against AMPK, AMPK (pThr¹⁷²), FAS, ACC (pSer⁷⁹), and ACC (cell signaling technology) and SREBP-1c, β-actin, and Lamin A/C (Santa Cruz Biotechnology). Consequently, the membranes were incubated with the corresponding horseradish peroxidase goat anti-rabbit IgG-HRP or anti-mouse IgG-HRP as secondary antibody (Santa Cruz Biotechnology). The immunoreactive proteins were detected with enhanced chemiluminescence (Pierce Biotechnology, USA). Band intensities were scanned (Epson Perfection V33) and quantified using the Image J software.