Ab181606

Proteins in H9c2 cells were extracted by lysis buffer (P0013J, Beyotime). After determining the concentration with a BCA detection kit (P0012, Beyotime), the proteins were loaded and electrophoresed on 10% SDS polyacrylamide gel (P0012AC, Beyotime), and then transferred onto PVDF membranes (FFP26, Beyotime,). After using 5% non-fat milk to block membranes for 60 min at 37°C, the following primary antibodies were incubated with membranes overnight at 4°C: Bcl-2 (ab59348, 1:1000, 26 kDa, Abcam, USA), Bax (ab32503, 1:1000, 21 kDa, Abcam), Cleaved caspase-3 (ab49822, 1:500, 17 kDa, Abcam), iNOS (ab3523, 1:200, 135 kDa, Abcam), Cox-2 (ab15191, 1:250, 69 kDa, Abcam), CDK4 (ab199728, 1:2000, 34 kDa, Abcam), Cyclin D1 (ab16663, 1:25, 36 kDa, Abcam), HSP70 (ab181606, 1:1000, 70 kDa, Abcam), and GAPDH (ab181602, 1:10000, 36 kDa, Abcam). Following extensive washing, protein bands were incubated with the secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, 42 kDa, Abcam) at 37°C for 2 h. The detection of signal was performed according to a standard ECL method (27), and analysis software (Image J 1.5i, National Institutes of Health, USA) was used for images to measured protein expression. GAPDH was used as housekeeping gene.

Five μm paraffin sections were treated with H₂O₂, washed in tris buffer saline, and incubated with rabbit anti-rat Hsp70 antibody (Heat shock protein 70, ab181606, Abcam, UK), a molecular chaperone that is expressed in response to stress. Nonspecific protein binding sites were blocked with normal goat anti-rabbit antibody (1 h), followed by the Avidin-Biotin Complex (1 h), DAB (10 min), and finally hematoxylin counterstained sections [46 ].

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).