T40104

Proteins were separated using 10% polyacrylamide denaturing gels and subsequently transferred onto polyvinylidene fluoride membranes by electroblotting. The primary antibodies employed included CORO1A (1:1000, TP71761, Abmart, China), CD3G (1:1000, T58478, Abmart, China), FCGR2A (1:1000, PHZ8459, Abmart, China), and GZMH (1:1000, PA1522, Abmart, China). The results were standardized against either GADPH (1:1000 dilution, M20024, Abmart, China) or β-Actin (1:1000 dilution, T40104, Abmart, China).

Cardiomyocytes were divided into four groups, lysates of cardiomyocytes were prepared in RIPA buffer, and protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, US). Equal amounts (30 μg) of proteins were separated on 10% SDS-PAGE (100 V for 120 min) and transferred into nitrocellulose membranes (120 min at 100 V) using standard procedures. The binding of nonspecific proteins to the membrane was blocked with blocking buffer (5% nonfat milk) at room temperature for 2 h. The membranes were probed with anti-CaMKII (ab52476, Abcam), anti-RIPK1 (ab106393, Abcam), anti-RIPK3 (sc-374639, Santa Cruz), and anti-β-actin (T40104, abmart) at 4°C overnight. After the membrane was washed three times with TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for 10 min each time at room temperature, the membrane was incubated with the second antibody (1 : 20000) for 1 h at 37°C. Then, the membrane was washed three times with TBST for 10 min each time at room temperature. Detection was performed using an ECL chemiluminescence kit (PK10003, Proteintech). Finally, the PVDF membrane was placed in the Bio-Rad ChemiDocXRS gel imaging system for photography under exposure, and band intensities were quantified using Image Lab software. Three independent replicates were conducted for this experiment.