Plates were immediately destained with four washes of ddH2O and photographed. Nuclear extracts were prepared as described.18 A consensus double-stranded Acalabrutinib mw HRE (Santa Cruz Biotechnology, Santa Cruz, CA) oligonucleotide was used for electrophoretic mobility shift assay (EMSA). End-labeling, oligonucleotide purification, and EMSA were

performed as described.19 A total of 30-50 μg nuclear extract was resolved on 10% polyacrylamide gels and transferred overnight to nitrocellulose. Membranes were blocked overnight with blocking buffer (5% bovine serum albumin in Tris-Borate-SDS with 0.01% Tween 20) with refrigeration, and subsequently probed overnight with anti–HIF-1α (R&D Biosciences) mouse monoclonal antibodies. Detection was

performed using anti-mouse horseradish-peroxidase–conjugated secondary antibody and chemiluminescent substrates. Band density was quantified using Labworks 4.0 image analysis. Statistical analysis was performed with Microsoft Excel using a two-tailed Student t test. P < 0.05 was considered significant. As has been reported elsewhere, ethanol feeding increased liver weight to body check details weight ratio, liver triglyceride, and serum ALT values and resulted in liver steatosis in WT mice compared with isocaloric diet-fed controls (Fig. 1A-E). To test our hypothesis that alcohol may increase the expression and activity of hypoxia-inducible factor-1, nuclear extracts from liver tissue

were evaluated for HIF-1 expression. We found that HIF-1α mRNA was up-regulated by ethanol feeding in WT mice (Fig. 1D). HIF-1α protein was also more abundant in alcohol-fed than in pair-fed livers (Fig. 2A,B). HIFs are primarily degraded by posttranslational hydroxylation and subsequent degradation of the alpha subunits by the ubiquitin/proteasomal system. To confirm that HIF-1α was transcriptionally active, we performed an EMSA using a commercially available HRE oligonucleotide. Our results showed a significant up-regulation of HIF DNA-binding activity in ethanol-fed animals versus pair-fed animals, suggesting HIF-1 activation (Fig. 2C,D). In order to determine the contribution of HIF-1α to ethanol-induced liver pathology, we used a mouse model Megestrol Acetate of hepatocyte-specific HIF-1α activation (HIF1dPA) described by Kim et al.10 Due to a mixed genetic background, Alb-Cre littermates that did not harbor the HIF1dPA transgene were selected as controls. To confirm the activation of HIF-1α in HIF1dPA mice, HIF-1α DNA-binding activity was examined in liver nuclear extracts from HIF1dPA and Alb-Cre control mice, and a significant up-regulation of HIF-1α DNA-binding activity was observed (P < 0.01; HIF1dPA pair-fed versus Alb-Cre pair-fed) (Supporting Fig. 1.) We found increased liver weight/body weight (LW/BW) ratios in HIF1dPA mice versus Alb-Cre controls even without alcohol feeding (Fig 3A).