Thbs1

The mouse heart tissues were homogenized in 0.5 ml of RIPA (1:1000 PMSF, Solarbio Science, Beijing) buffer using small tubes and vibrated every 10 min for 5 s at 4°C, and the above process was repeated four times. Solubilized proteins were centrifugated at 13,500 rpm for 25 min, and the supernatant was then collected. The protein concentration of each sample was quantified using the BCA Protein Assay kit (Beyotime, Shanghai). Protein lysates of each group were separated by electrophoresis with SDS-PAGE and electro-transferred onto a PVDF membrane (Millipore). Non-specific proteins on membranes were blocked with 5% non-fat dried milk for 2 h at room temperature, and the membranes were incubated overnight with the following primary antibodies (at a 1:1000 dilution, 4 °C): PRKG1, Ppm1k, Pltp, Hacd2, (ABclonal Technology, Wuhan, China); Pir (Abcam Technology, USA); Thbs1, (Cell Signaling Technology, Danvers, MA); Tmem70, β-tubulin (Santa Cruz Biotechnology). Then, the membranes were incubated with anti-mouse/anti-rabbit IgG antibody (ABclonal Technology, Wuhan, China) at a 1:10000 dilution for 1 h at room temperature. The specific complex was determined by an enhanced chemiluminescent (ECL) kit (Meilun, Dalian, China) and a multiplex fluorescent imaging system (ProteinSimple, CA).

Exosomes were lysed with RIPA buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.5% deoxycholate, 0.1% SDS, and 1.0% NP-40] containing protease inhibitor cocktail (Roche). Protein lysates (20 μg) were loaded onto a 4–20% Tris–glycine gradient gel (Biorad), transferred onto PVDF membranes and Western blotting was performed as per standard protocols. Following transfer, membranes were cut according to predicted molecular sizes and probed with following antibodies: THBS1 (Cell Signaling Technology, 37879), CD9 (Cell Signaling Technology, 13174), CD63 (System Biosciences, EXOAB-CD63A-1), Alix (Cell Signaling Technology, 2171), TSG101 (System Biosciences, EXOAB-TSG101-1) and Gelsolin (Cell Signaling Technology, 12953).