Comprehensive Western Blotting Protocol

Western blotting was performed using standard procedures as described previously30 (link). A lysis buffer was supplemented with a complete mini protease inhibitor (Roche), ALLN (25 μg/ml), 0.5 mM NaF, and 10 μM Na₃VO₄ just prior to use. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Bio-Rad). All samples (20 μg of protein) were suspended in lysis buffer, fractionated using NuPAGE 4%–12% Bis-Tris (Invitrogen) gels, and transferred to a Protran nitrocellulose transfer membrane (Whatman). The membrane was blocked using 1× phosphate-buffered saline (PBS) containing 5% non-fat milk for 1 h and incubated with a primary antibody [anti-ABCA1 (1:1000), anti-ABCG1 (1:1000), anti-AMPKα (1:1000), anti-SREBP-1 (1:250), anti-TF2B (1:1000), anti-β-actin (1:3000), anti-CREB (1:1000), anti-PCK1 (1:1000), anti-SRC1 (1:200), anti-G6Pase (1:200) or anti-CPT1a (1:1000)] overnight at 4°C. After washing with PBS–0.05% Tween 20 (0.05% T-PBS), the membrane was incubated with a secondary antibody (anti-rabbit, anti-mouse and anti-goat IgG HRP-linked; 1:2000) for 1 h at 4°C. The membrane was then washed with 0.05% T-PBS and detected with an ECL Western Blotting Detection Reagent (GE Healthcare) using an LAS-1000 system (Fuji Film).