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 then incubated for overnight with primary antibodies followed by 1 h incubation with peroxidase secondary anti-rabbit, anti-chicken and anti-goat antibodies (Millipore), respectively. Calnexin or β-actin was detected as a protein loading control. Chemiluminescence was detected by Image Reader LAS-4000 (Fijifilm) after adding Pierce EC Western Blotting Substrate (Pointe Scientific, Canton, MI, USA). Anti-CYP2E1 IgG was a gift from Dr. Jerome Lasker, Hackensack Biomedical Research Institute, Hackensack, NJ, anti-CYP2A5 IgG was a gift from Dr. Risto Juvonen, Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland. Anti-PPARα and anti-FGF21 IgGs were from Abcam, Cambridge, MA, USA. The other IgGs were from Santa Cruz Biotechnology, CA, USA. The bands of proteins were quantified with the Automated Digitizing System (ImageJ gel programs, version 1.34S; National Institutes of Health, Bethesda, MD).
