Ripa lysing buffer

The extraction of cell cytoplasmic and mitochondrial fractions were performed using the Mitochondria/Cytosol Fractionation Kit (BioVision, CA, USA) according to the manufacturer’s protocol. Total cell proteins were extracted by RIPA lysing buffer (Beyotime Institute of Biotechnology, China) and protein concentration was determined by the BCA Bradford protein assay kit (Beyotime Institute of Biotechnology, China). The extracted proteins were then mixed with loading buffer and boiled for 10 min. Equal amounts of proteins (40 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). After being blocked with 5% non-fat dry milk for 2 h, membranes were incubated overnight at 4 °C with primary antibodies against GAPDH (1:1000, Abcam, USA), VDAC (1:2000, abcam, USA), cytochrome C (1:500, Abcam, USA), Bcl-2 (1:500, Abcam, USA), Bax (1:800, Abcam, USA), cleaved caspase 3 (1:500, Abcam, USA), caspase 8 (1:500, Abcam, USA), cleaved caspase 9 (1:500, Abcam, USA), and then incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, China) for 2 h at room temperature. Membranes were visualized by an enhanced chemiluminescence (ECL) kit (Thermo, USA) and quantified by densitometry using an image analyzer (BandScan, USA).

The protein in U-EXO was collected using RIPA lysing buffer (Beyotime, China). Protein concentration was determined using a BCA kit (Beyotime, China). The CD9 and TSG101 protein expression in U-EXO were determined by western blot. Rabbit anti-human CD9 antibody (PROTEINTECH, USA) and rabbit anti-human TSG101 antibody (PROTEINTECH, USA) were used. HRP-labeled Goat Anti-rabbit IgG (Beyotime, China) was used as the secondary antibody.

Fresh heart tissues from mice were harvested and lysed using RIPA lysing buffer (Beyotime, Shanghai, China) to extract total protein. The primary antibodies were listed as follows: anti-GAPDH, anti-mTOR, anti-phosphorylated-mTOR (Ser2448) (p-mTOR), anti-p70S6K, anti-p-p70S6K (Thr389), anti-4EBP1, anti-p-4EBP1(Ser65), anti-Smad2, anti-p-Smad2 (Ser465/467), anti-Smad3, and anti-p-Smad3 (Ser423/425) (Cell Signaling Technology, USA); anti-TGF-β1 (Santa Cruz Biotechnology, USA); and anti-collagen I (Abcam, UK). Signals were detected using the FluorChem E data system (Cell Biosciences, USA) and then quantified using Quantity One 4.52 (Bio-Rad, USA).