Cardiac Protein Analysis Protocol

The cardiac tissue protein was extracted, and the protein concentration was assessed, as previously described (Ye et al., 2018 (link)). Protein was separated by electrophoresis using Laemmli sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to Immobilon-FL polyvinylidene fluoride (PVDF) membranes (Millipore, United States). Subsequently, after being blocked with 5% non-fat milk for 1 h, the membranes were incubated at 4°C overnight with the following primary antibodies: ChemR23 (Santa Cruz Biotechnology, United States), Bax [Cell Signaling Technology (CST), United States], Bcl-2 (Abcam, United States), c-caspase 3 (CST, United States), phosphorylated/total-p65 (p/T-P65; CST, United States), phosphorylated/total-extracellular signal-regulated kinase (p/T-ERK; CST, United States), phosphorylated/total-c-Jun N-terminal kinase (p/T-JNK; CST, United States), phosphorylated/total-p38 mitogen-activated protein kinase (p/T-P38 MAPK; CST, United States), and GAPDH (CST, United States). Then, the membranes were treated with a second antibody at room temperature for 1 h. Finally, antibody binding was detected with a two-color infrared imaging system (Odyssey, LI-COR Biosciences, Lincoln, United Kingdom). The protein expression intensity was normalized to that of GAPDH.