Hepatic proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After 1 h of blocking with 2% fat-free milk, membranes were incubated overnight with antibodies against CYP2E1, CYP2A5, acyl-CoA oxidase (AOX), alcohol dehydrogenase (ADH), PPARα, fatty acid synthase (FAS), Carnitine palmitoyltransferase I (CPT I), phosphorylated AMP-activated protein kinase (p-AMPK), total AMPK (T-AMPK), and calnexin or β-actin followed by 1 h incubation with peroxidase secondary anti-rabbit, anti-chicken, and anti-goat antibodies (Millipore). Calnexin or β-actin were detected as protein loading control. Anti-CYP2E1 IgG was gifted by Dr. Jerome Lasker, Hackensack Biomedical Research Institute, Hackensack, NJ; anti-CYP2A5 IgG was gifted by Dr. Risto Juvonen, Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland; anti-AOX was gifted by Professor Paul Van Veldhoven, K.U. Leuven, Belgium; finally, anti-p-AMPK and anti-T-AMPK were purchased from Cell Signalling Technology, and the remaining antibodies were from Santa Cruz Biotechnology, CA. Chemiluminescence was detected by Image Reader LAS-4000 (Fijifilm) after adding Pierce EC Western Blotting Substrate (Thermo Scientific). The bands of proteins were quantified with the Automated Digitizing System (ImageJ gel programs, version 1.34S; National Institutes of Health, Bethesda, MD).
