Hrp conjugated anti mouse or anti rabbit secondary antibody

Cryo-pulverized tissue from the I and R zones was lysed in RIPA buffer with protease inhibitors (Roche) then sonicated and spun at 12,000xg at 4° for 15 min. 10–30 ug of total protein was loaded into 4–15% Criterion TGX precast BioRad gels and ran in reducing conditions. Protein was transferred onto BioRad PVDF membranes, blocked in 3% non-fat milk or 3% BSA at room temperature for 2 h, then incubated overnight at 4 °C with primary antibodies for Serca2a (mouse monoclonal, 1:1000; Santa Cruz), IL-1β (mouse monoclonal, 1:1000; Cell Signaling Technologies) or GAPDH (rabbit polyclonal,1:7000; Sigma). Membranes were then washed and incubated with an HRP conjugated anti-mouse or anti-rabbit secondary antibody (BioRad) for 2 h at room temperature. Chemiluminescent blots were developed with Radiance ECL on an Azure c600 Western blot imaging system.
Because of the large number of animals in each group, not all samples could be loaded on the same gel. Two gels were, therefore, run that included samples from each of the four groups on each gel, having specific samples repeated on each gel. Signal intensity was then normalized between gels using the blot intensities of the repeated samples, as in our previous studies [29 ]. This method provides consistent normalization of all samples both within each group and across the minimum number of required gels.

sEV samples and cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer and separated by adding 40 μg protein on 7%, 10%, or 4–20% Mini-PROTEAN^® TGX™ Precast Gels, (BioRad, Hercules, CA, USA). The completed gels were transferred to a supported nitrocellulose membrane (BioRad). The membranes were blocked with 5% non-fat milk for one hour with gentle rocking. Membranes were incubated with primary antibodies overnight and then washed thrice for 10 minutes before addition of HRP conjugated anti-rabbit or anti-mouse secondary antibody (BioRad) for 1 hour. Membranes were subsequently washed and treated with ECL Western Blotting Substrate (Fisher Scientific) according to manufacturer’s instructions and antibodies used were either rabbit or mouse Isotype (Supplementary Table 8).

Exosome samples and cell lysates (prepared in RIPA buffer) were separated by adding 40 μg protein on 7%, 10%, or 4-20% Mini-PROTEAN^® TGX^™ Precast Gels, (BioRad, Hercules, CA) and transferred to a supported nitrocellulose membrane (BioRad). The membranes were blocked with 5% non-fat milk for one hour. Membranes were incubated with primary antibodies (Supplementary Table 3) overnight and washed thrice for 10 minutes before addition of HRP conjugated anti-rabbit or anti-mouse secondary antibody (BioRad) for 1 hour. Membranes were then washed and treated with ECL Western Blotting Substrate (Fisher Scientific) according to manufacturer's instructions.

Proteins from whole cell homogenization were isolated using lysis buffer (RIPA buffer supplemented with phosphatase and protease inhibitors). After a 30 min incubation at 4 °C and the subsequent centrifugation at 16,000× g for 30 min, the amount of proteins was quantified through Bradford assay. Fifty micrograms of proteins was loaded into SDS-polyacrylamide gels and transferred to PVDF membrane using a Bio-Rad Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were probed with the appropriate antibodies for PARP-1 (Abcam, dilution 1:5000), ꞵ-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, dilution 1:1000). As a secondary antibody, HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Bio-Rad, dilution 1:50.000) was used. For detection, Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare, Hatfield, UK) was applied before luminography).