Protease and phosphatase inhibitor cocktail solution

Proteins were isolated from rat hearts using lysis buffer (KeyGEN BioTECH, China) with a protease and phosphatase inhibitor cocktail solution (KeyGEN BioTECH, China). Thirty micrograms of protein was loaded on a 10% PAGE Gel Fast Preparation Kit (PG112, Epizyme Biotech, China). Proteins were separated and transferred to PVDF membranes (Millipore, USA). Membranes were incubated in TBST skim milk blocking buffer for 1 h at room temperature (RT) followed by primary antibodies overnight at 4 °C. The following primary antibodies were used: CD81 (sc-166,029, Santa Cruz, USA), Alix (sc-53,540, Santa Cruz, USA), MCU (26312-1-AP, Proteintech, USA), MICU1 (ab224161, Abcam, England), NCLX (ab83551, Abcam, England), PINK1 (23274-1-AP, Proteintech, USA), and GAPDH (AF7021, Affinity Biosciences, USA). Membranes were washed with TBST three times, incubated with secondary antibodies (701051, Zen Bioscience, China; ZB-2301, ZSGB-BIO, China) for 1 h at RT and washed with TBST three times. Membranes were probed using a chemiluminescence kit (Millipore, USA) and an Image system (Bio-Rad, USA).