Af0531

Total protein was extracted from myocardial tissues by using RIPA lysis buffer (Santa Cruz Biotechnology, Texas, USA). After the protein concentration was determined, equal amounts (20 µg) of protein were mixed with 5 × loading buffer. Then, the mixture was separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). After that, the membranes were incubated with 5% nonfat milk for 1 h at room temperature. Subsequently, the membranes were incubated with antibody working solutions containing CPT-1 (1:2000, DF12004, Affinity), GLUT4 (1:2000, AF5386, Affinity), AMPK (1:2000, AF6423, Affinity), p-AMPK (1:2000, AF3423, Affinity), PGC-1α (1:2000, AF5395, Affinity), NRF-1 (1:2000, AF5298, Affinity), mtTFA (1:2000, AF0531, Affinity), ATP5D (1:2000, 14,893, ptgcn) at 4 °C overnight. The next day, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were incubated with ECL reagent (Chengdu ZEN Bioscience Co., Ltd., Chengdu, China) to visualize the protein bands. The relative expression of target proteins was normalized to β-actin. Immunoblot band intensities were quantified using NIH ImageJ software.