Primary antibodies against gapdh

The total protein was extracted with RIPA (Beyotime, Shanghai, China) and the concentration was mesured by BCA (Beyotime, Shanghai, China). An equal amount of protein was resolved by SDS–PAGE and transferred onto PVDF membrane. Then, the membrane was incubated with the primary antibody and the second antibody respectively. As shown in Table S3, primary antibodies against Atrogin-1 (muscle atrophy F-box protein) and MuRF1(muscle RING finger-1) were purchased from Santa Cruz Biotechnology (CA, USA). Primary antibodies against GAPDH and PDK4 (pyruvate dehydrogenase kinase 4) were acquired from Proteintech (IL, USA). Antibodies against P-PDH, FoxO1 (forkhead box O1), PDH, ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase), P-HSL, and HSP90 were purchased from Cell Signaling Technology (MA, USA).

WB was performed according to standard procedures (BIO-RAD, Hercules, CA, USA). Briefly, cells with or without treatment were harvested and lysed with RIPA buffer (Beyotime) containing the proteinase inhibitor PMSF) (1 mM, Thermo Fisher Scientific). Protein concentration was determined using a BCA Protein Assay Kit (Beyotime). Equal amounts of protein were separated by SDS-PAGE (10%) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at RT for 2 h and incubated at 4 °C overnight with primary antibodies against GAPDH (1:8000; Proteintech) as a control, followed by horseradish peroxidase-conjugated secondary antibodies (Proteintech) for 1 h at RT. Primary antibodies including E-cadherin (1:1000; Cell Signaling Technology, CST, Boston, MA, USA), N-cadherin (1:1000; CST), Vimentin (1:1000; CST), Snail (1:1000; CST), TWIST (1:1000; CST), NLRP3 (1:1000; CST), ZEB1 (1:1000; CST), β-Catenin (1:1000; CST) and SMAD2/3 (1:1000; CST). Specific bands were detected using ECL chemiluminescence reagents (Millipore) and visualized using a Fusion Edge Multifunctional Imaging System (Vilber, France). Analyses of the bands were performed using ImageJ software (V1.8.0.112).