Anti cleaved caspase 8

Total myocardial protein from the apex of the left ventricle was extracted using protein lysis buffer, and the Lowry method was used to determine the protein concentration. Equal amounts of protein (50 µg) were fractionated by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, Mass, USA). The membranes were blocked for 1 h at room temperature using 5% non-fat milk in Tris-buffered saline supplemented with Tween-20 (TBST) and incubated overnight at 4°C with the following primary antibodies: anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved caspase-8 and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; all obtained from Abcam, Cambridge, Mass, USA). The membranes were washed with TBST and incubated with goat anti-rabbit IgG HL secondary antibodies (1: 10000 Abcam, Cambridge, Mass, USA) at room temperature for 2 h. The immuno-reactive bands were detected by exposing the blots in an Odyssey infrared imaging system (LI-COR). GAPDH was used as the internal control.

Proteins were extracted from cell lysates using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) and the protein concentration was measured using the Enhanced BCA Protein Assay Kit (Beyotime) according to the manufacturer’s instructions. Heat-denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes, followed by blocking with 1% bovine serum albumin solution for 1 h. The membranes were then incubated with primary antibodies, including anti-HMGB1 (1:1000; Abcam), anti-TLR4 (1:1000; Abcam), anti-NF-κB p65 (1:1000; Abcam), anti-cleaved caspase-8 (1:1000; Abcam), anti-cleaved caspase-3 (1:1000; Abcam), anti-GAPDH (1:1000; Zhongshan Jinqiao Biotechnology, Beijing, China), and anti-Lamin A (1:1000; Abcam) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (1:4000; EarthOx, Millbrae, CA) at room temperature for 30 min. Protein bands were detected using an enhanced chemi-luminescence kit for western blotting (Beyotime) according to the manufacturer’s instructions. GAPDH or Lamin A was used as an internal control. Relative densitometry was calculated using Image J2x analysis software (National Institutes of Health, Bethesda, MD).