Ox camkii

Heart samples were obtained after reperfusion for 2 h and were immediately stored at -80°C before western blot analyses were performed. Protein was extracted using the RIPA protein lysate (ASPEN, Wuhan, China), and the concentration of the protein was determined using the BCA protein concentration assay kit (ASPEN, Wuhan, China). Subsequently, protein (40 μg) was separated by 10% SDS-PAGE and electrotransferred to PVDF membranes. After incubation with corresponding antibodies, the PVDF membranes were then washed by TBST and incubated with a secondary antibody for 1 h at room temperature. A chemiluminescence method was used to detect protein bands, and AlphaEaseFC software processing system (Alpha Innotech, USA) was used to analyze the optical density of target bands. The following primary antibodies were used for western blot analysis: Bax (1 : 2000), p-ASK1 (1 : 500), ASK1 (1 : 1000), NF-κB p65 (1 : 1000), histone H3 (1 : 3000), p-JNK (1 : 1000), and JNK (1 : 2000) (Cell Signaling Technology, USA); Bcl-2 (1 : 2000), caspase 3 (1 : 2000), p-CaMKII (1 : 1000), CaMKII (1 : 500), TNF-α (1 : 1000), IL-6 (1 : 500), IL-1β (1 : 500), and RyR2 (1 : 500), GAPDH (1 : 10000) (Abcam, UK); ox-CaMKII (1 : 500) (GeneTex, USA); p-RyR2 (Ser2814, 1 : 500) (Badrilla, UK); and cleaved caspase 3 (1 : 1000) (Affbiotech, USA).

Total proteins were isolated from cultured cells by using a lysis buffer. Then equal amounts of protein lysates were separated by SDS-page gel electrophoresis. After transferring onto a PVDF membrane, protein was incubated with primary antibodies against Fgf21, Sirt1, Troponin I, t-CamKII, Calpian 1, NOX2, NOX4, GAPDH (Abcam), Nrf2, MHC, SOD2, UCP3, p-RyR2, β-Klotho (Abclonal), ox-CamKII (GeneTex), RyR2, FgfR1 (Proteintech), L-type calcium channel α1C-subunit (LCC) (Affinity), α-SMA, Collagen-1A1, CTGF, and Tubulin (Servicebio). Then they were incubated with a secondary antibody conjugated to HRP (1:5,000) for 1 h. Signals of proteins were identified by chemiluminescence and quantified by densitometry.