Ab125011

The sEV-enriched supernatant was denatured in 5× sodium dodecyl sulfonate (SDS) buffer and used for Western blotting (10% SDS-polyacrylamide gel electrophoresis; 10–30 µg protein/well). Antibodies against the following were used: CD63 (sc-5275; Santa Cruz Biotechnology, Dallas, TX, USA), HSP70 (ab181606; Abcam, England), TSG101 (ab125011; Abcam, England), and calnexin (10427-2; Proteintech, Rosemont, IL, USA). The wet rotation method was used, and the membrane was completely immersed in 3% bovine serum albumin (BSA)-TBST and gently shaken at room temperature for 30 min. The primary antibody was diluted with 3% bovine serum albumin (BSA)- Tris-buffered saline Tween (TBST), incubated at room temperature for 10 min, and placed at 4 °C overnight. On the next day, the membrane was incubated at room temperature for 30 min, followed by washing with TBST 5 times for 3 min each time, followed by incubation with the secondary antibody. Electrogenerated chemiluminescence reagents were added to the membranes, and signals were detected by an automatic chemiluminescence imaging system (Tanon 4600; Tanon Co., Ltd., Shanghai, China).

Exosome-specific markers, including positive (CD9, CD63, and TSG101) and negative markers (GRP94), were used to identify exosomes by western blotting. Total proteins (25 µg) in the extracted resuspension of exosomes were sequentially subjected to gel electrophoresis (10% SDS-PAGE), membrane transfer, blocking, incubation with primary antibodies specific for CD9, CD63, TSG101, and GRP94 (ab92726, ab134045, ab125011, ab238126, Abcam, Cambs, UK), incubation with the goat anti rabbit secondary antibodies, and enhanced chemiluminescence (ECL) to examine exosomal protein expression (22 (link), 23 (link)).

Tissues were lysed in RIPA buffer containing a protease inhibitor cocktail (Beyotime Institute of Biotechnology, Haimen, China). Total protein isolated from the myocardium was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in Tris-buffered saline with 0.1% Tween (TBST) containing 5% nonfat dry milk for 1 h at RT and then incubated in universal antibody diluent (New Cell & Molecular Biotech, Suzhou, China) using the appropriate primary antibody overnight at 4°C. The primary antibodies used in this experiment were specific to the following antigens: iNOS (1 : 1000; ab210823; Abcam, Cambridge, UK), Arg1 (1 : 1000; ab91279; Abcam, Cambridge, UK), C/EBPα (1 : 1000; 18311-1-AP; Proteintech, Chicago, USA), PU.1 (1 : 2000; ab88082; Abcam, Cambridge, UK), SOCS1 (1 : 1000; YT4362; Immunoway, Newark, USA), CD63 (1 : 1000; ab213090; Abcam, Cambridge, UK), HSP70 (1 : 1000; ab2787; Abcam, Cambridge, UK), and TSG101 (1 : 1000; ab125011; Abcam, Cambridge, UK). The membranes were incubated with HRP-conjugated secondary antibodies after washing in 5% TBST. Finally, the protein bands were detected using a gel chemiluminescence imaging analysis system and the Immobilon Western Chemiluminescent HRP Substrate reagent (Millipore, Billerica, USA) and then were analyzed using ImageJ.

EV isolation and characterization by IB was carried out as reported earlier [47 (link), 48 (link)]. Blocked membranes were probed with either rabbit monoclonal antibodies (mAbs) to TSG101 (ab125011) from Abcam (Cambridge, UK) and CD63 (55051) and FAK (13009) from Cell Signaling Technologies (Danvers, MA, USA) or mouse mAbs to human CD81 (ab23505) from Abcam, Alix (sc53540) and human CD9 (sc13118) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and human SERPINA1 (NBP2-52557) from Novus Biologicals (Littleton, CO, USA).