Anti pparα

RIPA buffer containing PMSF and phosphatase inhibitors was used to extract the protein of MCMs (Beyotime Biotechnology Co., China). Protein concentrations were determined using the BCA protein assay kit (Beyotime Biotechnology Co., China) according to the manufacturer’s instructions. The samples were stored at −20°C for further use. The protein samples of each group were separated by 8% or 12% SDS-PAGE and transferred to PVDF membranes (MerckMillipore Co., USA). The PVDF membranes were sealed with rapid blocking solution at room temperature. Western blotting analysis was performed using anti-P-mTOR (1:1000; Cell Signaling Technology Co., USA), anti-mTOR (1:1000; Proteintech Group Co., China), anti-PPARγ (1:1000; Santa Cruz Biotechnology Co., USA), anti-PPARα (1:1000; GeneTex Co., USA), anti-PGC-1α (1:1000; Abcam Co., UK), anti-UCP2 (1:1000; Abmart Biotechnology Co., China), anti-BNP (1:1000; Abclonal Biotechnology Co., China), and anti-GAPDH (1:5000; Proteintech Group Co., China) antibodies. Anti-mouse or anti-rabbit horseradish peroxidase-labeled antibodies (1:5000; Proteintech Group Co., China) were used for detection. Membranes were revealed with ECL chemiluminescent substrate (Advansta Co., USA). Finally, the fusion imaging system was used to detect the relative densities of the bands.