Bs 20669r

The cells were lysed by lysis buffer (Boster, Wuhan, China) containing 1 mM protease inhibitor PMSF (Boster, Wuhan, China). To obtain total protein, the lysate was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was the total protein. The total protein was denatured and then separated by SDS-PAGE (8–15%). The separated protein was transferred to a nitrocellulose membrane (Univ-bio, Shanghai, China). The membrane was blocked with a protein free quick blocking solution (Beyotime, Shanghai, China) for 15 min and incubated with specific primary antibodies for 3 h at room temperature. The membrane was washed for 3 times with Tris-buffered saline with 1% Tween 20 (TBST) (Beyotime, Shanghai, China) and incubated with secondary goat anti-rabbit IgG (1:5000, BA1056, Boster, Wuhan, China) for 1 h at room temperature. The membrane was washed again for 3 times with TBST, and the blot was detected by enhanced chemiluminescence solution (Meilune, Dalian, China) and analyzed by Image J (version 1.4.3.67) software. The primary antibodies used are as follows: β-actin (1:5000, bsm-33036M, Bioss, Beijing, China), GRP78 (1:1000, bs-1219R, Bioss, Beijing, China), CHOP (1:500, bs-20669R, Bioss, Beijing, China), STIM1 (1:500, A9764, Abclonal, Wuhan, China), ORAI1 (1:500, A7412, Abclonal, Wuhan, China).

Total proteins isolated from the heart and cardiomyocytes were quantified by bicinchoninic acid (BCA) (Beyotime Institute of Biotechnology, Haimen, China) and subjected to Western blotting. Briefly, equal amounts of proteins were boiled for 5 min with loading buffer and then separated by SDS-PAGE. The proteins were transferred to PVDF membranes (EMD Millipore, Bedford, MA, USA) and blocked with buffer (5% non-fat milk in TBST buffer) for 1 h. The PVDF membranes were incubated with primary antibody against glucose-regulated protein 78 (GRP78) (D151791; Sangon Biotech, Shanghai, China; 1:500 dilution), CHOP (bs-20669R; Bioss, Beijing, China; 1:500 dilution), B-cell lymphoma 2 (Bcl-2; BA0412; Boster, Wuhan, China; 1:400 dilution), Bcl-2-associated X protein (Bax; D120073; Sangon Biotech; 1:500 dilution), endoplasmic reticulum oxidoreductase 1α (Ero1α; bs-10551M; Bioss; 1:500 dilution), Ero1β (bs-14627R; Bioss; 1:500 dilution) or protein disulfide isomerase (PDI; bs-4250R; Bioss; 1:500 dilution). After being washed with TBST buffer, the membranes were incubated with corresponding secondary antibody (A0208 and A0216; Beyotime Institute of Biotechnology; 1:5,000 dilution) at 37°C. After being washed, the bands were developed using ECL regent (Beyotime Institute of Biotechnology) and quantified by Media Cybernetics Gel-Pro analyzer. β-actin was used as an internal control.