Cocktail of protease and phosphatase inhibitors

The methods of protein extraction and Western blot were performed as previously described by Chiu et al. [11 (link)]. Briefly, the radioimmunoprecipitation assay (RIPA) buffer with a cocktail of phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) was used for liver protein extraction. A BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure protein concentration. For immunoblotting, 50–100 μg of proteins were separated through 8–12% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking using 5% non-fat dry milk solution for 1 h, membranes were probed with primary antibodies specific for phosphorylated AMP-activated protein kinase α (p-AMPKα; #2531; 1:1000), AMPKα (#2532; 1:1000) (Cell Signaling Technology, Danvers, MA, USA), PPARγ (sc-7273; 1:1000), and GAPDH (sc-47724; 1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). An Enhanced Chemiluminescence kit (Bio-Rad) was used to detect the target protein expression, which was exposed to a Fujifilm X-ray film (Tokyo, Japan). The densitometry of target protein was analyzed by the Image J 1.8 software (National Institutes of Health, Bethesda, MD, USA).