2.6. Western blot analysis

Western blot was performed as previously described by Xu et al.43 In brief, total protein was extracted from the liver tissues using a protein extract kit according to the manufacturer's protocol. All protein concentrations were measured with BCA protein assay. A total of 20 μg of protein from each sample were separated by a 12% SDS‐PAGE and transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% (w/v) non‐fat milk for 2 hours followed by incubated with primary antibodies (Nrf2, Keap1, AMPK, GSK3β, Beclin1, LC3, NLRP3, ASC, Casapase‐1, IL‐1β, p‐p65/p65, p‐IκBα/IκBα, Shh, Smo, Gli1 and β‐actin) and secondary antibody (anti‐rabbit IgG and anti‐mouse IgG). Lastly, the membranes were visualized with the enhanced chemiluminescence (ECL) reagent in Western blotting analysis system with Image Lab (Bio‐Rad).