Western blot analysis of liver tissue

Following previously described procedures,⁽⁷⁾ the homogenized tissues were centrifuged at 15,000 rpm for 30 min and the supernatants were stored at −70°C until further analysis. Aliquots of tissue homogenates were used for protein assay (Bio-Rad protein assay reagent) and Western blot analysis. The liver homogenates were probed for sterol regulatory element-binding protein-1 (SREBP-1), stearoyl-CoA desaturase 1 (SCD-1), fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), low-density lipoprotein receptor (LDLR), and cholesterol-7α-hydroxylase (CYP7α1), and peroxisome proliferator-activated receptor-α (PPARα) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500 dilution) and IgG conjugated antibody (Santa Cruz Biotechnology; 1:10,000 dilution). The relative expression of those proteins in each tissue was quantified by densitometric scanning of the western blots using Image-pro plus software (Media Cybernetics, MD).