Ecl luminescent substrate

The proteins were extracted and separated using the Epizyme (#PG112) kit before being transferred to a PVDF membrane (Epizyme, #WJ001). For 10 min, a protein-free rapid-blocking solution (Epizyme, #PS108) was used to block the cells. The primary antibody was incubated at 4°C overnight before being rinsed three times for 10 min with Tris-buffered saline containing Tween 20 (TBST). The secondary antibodies were then incubated at room temperature for another 1 h. The Bio-Rad X-ray development system was used to obtain the results after the ECL luminescent substrate (Tanon, #180-501) was added dropwise to the membrane (Bio-Rad, CA, USA). The loading control was -Actin, and the data were analyzed using Image J software. The antibodies used for Western Blot in this study are listed in Supplementary Table 1.

Left ventricular tissue or cell samples were collected and lysed by RIPA buffer (720 μL of RIPA buffer, 20 μL of phenylmethylsulfonyl fluoride, 100 μL of complete protease inhibitor cocktail, 100 μL of Phos-stop, 50 μL of NaF, and 10 μL of Na3 VO4 in a final volume of 1 mL). After lysis and centrifugation, the supernatant was taken as total protein and quantified by a BCA protein kit (Pierce). After separation by using SDS polyacrylamide gel electrophoresis, the proteins were transferred to a 0.45 μm PVDF (IPVH00010, Millipore) membrane. The membrane was then blocked with 5% nonfat milk at room temperature for 1 h. The PVDF membrane was cleaned three times with TBST, 5 min each time. A primary antibody was added and incubated at 4 °C overnight. The secondary antibodies were added the next day. ECL luminescent substrate (1705062, Bio-Rad) was used for imaging and the Bole gel imaging system (ChemiDoc XRS+) was used for signal collection. Image Lab (Version5.1) software was used to analyze the results, and the corresponding antibody information is Supplementary Table 1.