Polyvinylidene uoride membrane

Liver tissue was lysed with RIPA lysis buffer to extract total protein. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scienti c). Then, 20 µg of protein were separated on sodium dodecyl-sulfate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene uoride membranes (Millipore, Burlington, MA, USA). The membrane was blocked with 5% non-fat milk for 1 h and then incubated with speci c primary antibodies overnight at 4 ℃ (anti-BMAL1:1:1000, ab235577; anti-cluster of differentiation 36 [CD36]: 1:1000, ab252922; anti-peroxisome proliferator-activated receptor gamma [PPARγ]: 1:1000, ab272718; anti-glyceraldehyde-3-phosphate dehydrogenase [i.e., GAPDH]: 1:1000, ab8245; and anti-β-ACTIN:1:1000, ab8226; all purchased from Abcam, Cambridge, UK). After incubation, the membrane was washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000, ab288151, purchased from Abcam, Cambridge, UK) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence substrate (Thermo Fisher Scienti c). Protein band density was quanti ed using ImageJ software (National Institute of Health, Bethesda, MD, USA) and normalized to β-actin levels.

Western blot analysis was performed as described previously [26] . In brief, the protein concentration was measured using a bicinchoninic acid protein assay kit (YTHXBio, China). The extracted proteins were then separated by electrophoresis using a 12% sodium dodecyl sulphate polyacrylamide gel. The proteins were transferred to polyvinylidene uoride membranes (Millipore, USA) and blocked using Tris-buffered saline with Tween buffer containing 3% BSA. The membranes were incubated with primary antibodies against Bax (1:500), phosphorylated AMPK (Thr 172) (1:1000), total AMPK (1:5000) (all from Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:5000; Abcam), and GADPH (1:10000; YTHXBio, China) at 4°C overnight. The membranes were washed three times and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000) at 37°C for 40min. Finally, the bands were detected by an enhanced chemiluminescence kit (Millipore, USA) and the results were quanti ed using Total Lab Quant V11.5 (Newcastle upon Tyne, UK). The levels of Bcl-2/Bax (anti-/pro-apoptotic) proteins and AMPK-p/AMPK proteins were also normalized to that of GAPDH.