Tsg101

ADSC-EVs, ADSC-NVs, cells, or tissues were lysed using RIPA buffer (Beyotime Biotechnology, P0013B) containing 1% PMSF (Aladdin, P105539), and the supernatant from lysates was used for subsequent western blot analysis. Protein samples were resolved by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore, IPVH00010). After blocking in TBST with 5% non-fat milk for 2 h, membranes were incubated at 4 °C overnight with the following primary antibodies: CD29 (Novus, NBP2–36561), TSG101 (Servicebio, GB11618), ANG1 (Proteintech, 23,302–1-AP), VEGF (Boster Bio, PB0084), PTEN (Proteintech, 22,034–1-AP), PI3K (Proteintech, 60,225–1-AP), Akt (Affinity, AF6261), and p-Akt (Affinity, AF0016). On the next day, membranes were washed with TBST and hybridized with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Boster Biological Technology, BA1054) and anti-mouse (Boster Biological Technology, BA1051) IgG antibodies for 2 h at room temperature. Finally, bands were visualized using ECL blotting detection reagents (Servicebio, G2014).

Exosome samples were homogenized in RIPA lysis buffer with protease inhibitor cocktail and PMSF, and vortexed every 5 min for 30 min to lyse the exosomes on ice. Subsequently, lysates were centrifuged at 12,000g for 1 h, and supernatants were collected and stored at -80°C for later analysis. Protein concentration was determined by the BCA assay (Thermo Fisher Scientific). Protein was electrophoresed in 8-16% Tris-HCl polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad) for 90 min at 4 °C. After 1 h in TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk, membranes were incubated overnight with primary antibodies against CD63 (1:1000; Biolegend), HSP70 (1:1000; Cell Signaling Technology), TSG101 (1:200; Servicebio), CD9 (1:500; Bioss), Calnexin (1:1000; Santa Cruz). After rinsing, blots were incubated with peroxidase-linked secondary antibodies, treated with ECL substrate, and signals were visualized using BioRad.

We extracted proteins from 30 mg of canine atrial tissue or cultured cells. The protein concentration was determined by a BCA Protein Assay Kit (Aspen, as1086) according to the manufacturer's instructions. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to a 0.45 μM PVDF membrane by semidry transfer. The PVDF membranes were then blocked with 1% polyvinylpyrrolidone-40 and 0.05% Tween-20 for 30 minutes. The expression of target proteins was determined by incubating the membranes with the following primary antibodies overnight at 4°C: GAPDH (Servicebio, China, 1 : 1000), CD81 (Abmart, China, 1 : 1000), Rab27a (Servicebio, China, 1 : 1000), KCa3.1 (Proteintech, China, 1 : 1000), TSG101 (Servicebio, China, 1 : 1000), AKT (Servicebio, China, 1 : 1000), and p-AKT (Abmart, China, 1 : 1000). Visualization was performed on a chemiluminescence system after incubation with horseradish peroxidase-conjugated secondary antibodies (Proteintech, China, 1 : 3000) at room temperature.