P ask1

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).

Protein was extracted from liver tissue or cell cultures as described.^{2 (link)} Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). Monoclonal rabbit anti-mouse NICD, HSF1, Snail, TRX1, TXNIP, NLRP3, ASC, cleaved caspase-1, p-ASK1, ASK1, Bcl-2, Bcl-xL, cleaved caspase-3, and β-actin Abs (Cell Signaling Technology, MA) were used. The membranes were incubated with the Abs and then developed according to the Pierce SuperSignal West Pico Chemiluminescent Substrate protocol (Pierce Biotechnology, Rockford, IL). The relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY) and expressed in absorbance units (AU) by comparison with β-actin expression.