Cells were lysed in SDS Laemmli buffer (Bio-Rad, Hercules, CA, USA) supplemented with 1% protease inhibitor cocktail, 200 uM sodium orthovanadate and 5% β-mercaptoethanol (all from Sigma-Aldrich Corp., St. Louis, MO, USA). Lysates were heated at 95°C for 10 minutes, separated by SDS-PAGE on 4% to 20% gradient or 10% Tris-glycine precast gels (Life Technologies), and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Waltham, MA, USA). Rabbit or mouse antibodies specific for phospho (p)-AKT (Ser473), AKT, p-FOXO1 (Ser256), FOXO1, β-actin (all from Cell Signaling Technology, Danvers, MA, USA), and the precursor form of SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used. Membranes were blocked with 5% bovine serum albumin (for antibodies of p-AKT and p-FOXO1; Sigma-Aldrich Corp.) in Tris-buffered saline containing 0.01% Tween-20 (TBS/T) or with 5% skim milk in TBS/T (for antibodies AKT, FOXO1, β-actin and SREBP-1). All primary antibodies were diluted 1:1000 in blocking buffer except for p-AKT (1:4000), SREBP-1 (1:200), and β-actin (1:10,000). HRP-conjugated secondary antibodies were goat anti-rabbit IgG and Fc-specific goat anti-mouse IgG diluted 1:5000 (Sigma-Aldrich Corp.). Proteins were visualized with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL, USA) using a G-Box gel documentation station (Syngene, Frederick, MD, USA). Image analysis and densitometry were performed using GeneSys software (Syngene).