Ab93628

Cultured cells were lysed with RIPA lysis buffer containing 1% protease inhibitor cocktail (Beyotime, China). The protein concentration in the lysates was measured using a BCA protein assay kit (Beyotime, China). Protein samples were separated by 10% SDS-polyacrylamide gel and then transferred to polyvinyl difluoride membranes (Millipore, USA), which were then blocked for 1 h with 5% skim milk in TBST (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20 (pH8.3)) at room temperature. The membranes were incubated overnight at 4 ℃ with anti-ASIC1a antibody (Ptoteintech, 27235-1-AP, China) or anti-NFATc2(Abcam, ab92490, UK), anti-NFATc1 (Abcam, ab177464, UK), anti-NFATc3 (Abcam, ab93628, UK), anti-NFAT5 (Abcam, ab137407, UK), anti-NFATc4 (CST, 2188, USA), anti-Na⁺/K⁺-ATPase (Abcam, ab76020, UK), anti-H3 (Abcam, ab1791, UK), anti-β-actin (ZSGB Bio, TA-09, China) antibodies. The membranes were washed in TBST and incubated with secondary antibody (1:5000, ZSGB Bio, China) for 1 h at room temperature followed by exposure to electrochemiluminescence. The results were expressed as a percentage of control signals in each blot to correct for variations between blots.

Cells and fresh myocardial tissues from the myocardial infarction (MI) area were washed three times with cold PBS and lysed with the RIPA buffer (Beyotime, P0013B) containing protease and phosphatase inhibitors (Boster, AR1183) according to the manufacturer’s instructions. A BCA protein assay kit (Beyotime, P0011) was used to determine the protein concentration. Equal amounts of protein were loaded on the wells of each line in a 10% SDS-PAGE gel for electrophoresis and then transferred to a PVDF membrane. The membranes were then blocked with 5% BSA in TBST buffer for 1 h at room temperature, and incubated with a primary antibody against caspase-3 (CST, 9662s), BAX (CST, 2774), BCL-2 (Abcam,196495), OCT4 (Boster,bs-0830R), p-AKT (CST, 13038S), AKT (CAT, 2920), ERK1/2 (CST, 9102), p-ERK1/2 (CST, 4370S), p-β-catenin (ser33/37/T41, CST, 9561s), β-catenin (Santa, sc7199), SFRP2 (Abcam, ab86329), and NFAT4 (Abcam, ab93628) at 4 °C overnight. Following three washes with TBST, the membranes were incubated with their corresponding secondary antibody (Jackson 111-035-003 and Jackson 111-005-003) at room temperature for 1 h, and the blots were visualized using chemiluminescence (ECL; Forevergen Biosciences Center, Guangzhou, China). Blots were quantified by densitometric scanning. The level of protein expression was normalized against GAPDH controls.