Western blotting analysis.

Mouse hearts (atrial tissues were removed) were snap frozen in liquid nitrogen and stored at –80°C until use. Tissue were homogenized in RIPA buffer (Beyotime Biotechnology, P0013B) supplemented with PMSF and protease inhibitor cocktails (Roche, 11873580001). Equal amounts of sample proteins were loaded onto SDS polyacrylamide gels, separated by a vertical electrophoresis system, and finally transferred to a PVDF membrane (EMD Millipore). Afterward, the membranes were blocked in 5% BSA in Tris-buffered saline with Tween detergent (TBST; 150 mM NaCl, 50 mM Tris, and 0.5 nM Tween 20 at pH 7.5) for 1 hour and incubated with primary Abs in 5% BSA overnight at 4°C. The following day, the membranes were rinsed 3 times in TBST for 5 minutes each, incubated with secondary Abs at room temperature for 2 hours, rinsed 3 times in TBST, and finally visualized using ECL. Quantification analysis of protein levels was performed using ImageJ software (NIH).