Tissue samples were subjected to immunoblotting analysis as described previously [15 (link)]. Briefly, liver tissue was homogenized in lysis buffer and centrifuged at 13,000 rpm, 4°C for 15 minutes. The total concentration of protein was measured using the Bradford protein assay dye (Bio-Rad Laboratories, Hercules, CA, USA). The whole lysate was mixed with 5× sample buffer and denatured at 95°C for 6 minutes. The total proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (GE Healthcare BioSciences Co., Piscataway, NJ, USA). After protein blocking, the membranes were incubated with anti-phospho-nuclear factor kappa B (p-NF-κB, 1:1,000, #3033, Cell Signaling Technology), anti-total-nuclear factor kappa B (t-NF-κB, 1:1,000, #8243, Cell Signaling Technology), anti-acyl-CoA oxidase 1 (ACOX1; 1:1,000, sc-98499, Santa Cruz Biotechnology Inc.), and anti-β-actin (1:3,000, A5326, Sigma-Aldrich) antibodies. The blots were reacted with peroxidase-conjugated secondary antibodies (Vector Laboratories Inc., Burlingame, CA, USA), and the positive immunoreactive protein bands were detected using LAS-3000 film (FUJIFILM Corporation, Tokyo, Japan). All protein levels were normalized to β-actin.