Omni ecltm femto light chemiluminescence kit

Protein extracts were extracted from liver tissues or HepG2 cells using the Protein Lysis Kit (Beyotime, China) and then separated on 12% SDS-PAGE gels. Then, gels were transferred to polyvinylidene difluoride (PVDF) membranes and PVDF membranes were incubated with primary antibodies against AdipoR1, AdipoR2 (Affinity Biosciences, China), AMPK, phosphorylated-AMPK (p-AMPK), LKB1, phospho-LKB1 (p-LKB1) (Cell Signaling Technology, MA, USA), SIRT1, nuclear factor erythroid-2-related factor 2 (Nrf2), carnitine palmitoyltransferase-1A (CPT1A), silent information regulator transcription 3 (SIRT3), PGC1α and GAPDH (Proteintech Group, USA) overnight at 4 °C. The PVDF membranes were washed and incubated with secondary antibodies at room temperature for 1h. Finally, Omni-ECL^TM Femto Light Chemiluminescence Kit (EpiZyme, China) was used to detect specific protein expression. The images were obtained using Chemi-Lumin One Ultra (Tanon, China).

Briefly, cell extracts were prepared using cell lysis buffer for Western and IP (Beyotime, Wuhan, China). The protein concentrations in the supernatant were measured using the bicinchoninic acid test (CWBIO, Beijing, China). The same amounts (20 μg) of total protein were loaded into SDS-PAGE gels (EpiZyme, Shanghai, China) for electrophoresis. After that, separated proteins were electroeluted at 120 volts onto PVDF membranes (Roche Applied Science, Germany). The membranes were blocked with Protein Free Rapid Blocking Buffer (Shanghai Epizyme Biomedical Technology Co., Ltd, China) for 15 min and incubated with primary antibodies (exhibited in Table S3) at a dilution of 1:1000 for 24 h at 4 °C, followed by HRP-conjugated secondary antibodies incubation for 2 h at RT. Finally, bands were visualized using the Omni-ECL^TM Femto Light Chemiluminescence Kit (EpiZyme, Shanghai, China) and imaged by the Syngene G: BOX Chemi XT4 system.