Ethanol administration activates the transcription factor NF-κB and increases expression of mRNA for the inflammatory cytokines TNF-α and IL-6 in wild-type but not NADPH oxidase–deficient mice. (a) NF-κB DNA binding activity in the liver was assessed by electrophoretic mobility shift assay using whole liver nuclear extracts from wild-type mice fed high-fat control (lane 1) or ethanol-containing (lane 2) diet, or p47^phox–/– mice fed high-fat control (lane 3) or ethanol-containing (lane 4) diet. No binding was detected with no nuclear extract added (lane 5). Nuclear extracts from wild-type mice fed ethanol-containing diet (same as in lane 2) were used for competition experiments (200-fold excess of the unlabeled oligonucleotide, lane 6) and supershift experiments (p50 or p65 anti-serum, lanes 7 and 8, respectively). Representative data from four separate experiments are shown. (b) Data shown are results of densitometric analysis of the NF-κB/DNA complex images. Density of the NF-κB/DNA complex image in livers from wild-type mice fed high-fat control diet was set to 100%. Data represent mean ± SEM (n = 4). CON, high-fat control diet; ETH, high-fat ethanol-containing diet. ^AP < 0.05 compared with wild-type mice fed high-fat control diet, ^BP < 0.05 compared with wild-type mice fed ethanol-containing diet by two-way ANOVA with Bonferroni’s post-hoc test. (c) Total mRNA was prepared from livers of wild-type or p47^phox–/– mice administered high-fat (CON) or ethanol-containing (ETH) diets for 4 weeks, and RNase protection assays were performed. Representative data from four separate experiments are shown.