Image 4

After extraction from cells, the protein concentration was determined with the BCA Protein Assay Kit (Beijing ComWin Biotech Co., Beijing, China). Western blotting was performed according to the manufacturer’s instructions. The proteins on the membranes were detected using ECL and visualized at Bio-Rad Laboratories (Berkeley, CA, USA). The western blots are representative of at least three independent experiments. Densitometric analysis of each band for the target protein was performed with Scion Image 4.03.

The left ventricles of the remaining rats were weighed to calculate the ratio of left-ventricular weight to body weight. The size of the infarct was obtained by calculating the percentage of the infarcted area against the whole LV area using a digital imaging program (Scion Image 4.03, Bethesda, MD, USA). The tissues from the autopsy specimens were embedded in paraffin or frozen for cryostat sectioning and were then stained by hematoxylin and eosin or used in immunofluorescence assays. For immunocytofluorescence, cells were fixed with fresh 4% paraformaldehyde in PBS.

The TLR4, MyD88, and NF‐κB protein levels in lysed cell were examined by western blot analysis. Protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, Jiangsu, China). Total protein (20 μg) was fractionated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 10% defatted milk powder solution at room temperature for 2 hr and incubated overnight at 4°C with the rabbit antibodies against TLR4 (concentrate on 1:500), MyD88 (concentration 1:700), NF‐κB (concentration 1:700), GAPDH (concentration 1:10,000), and beta‐actin (concentration 1:2,000). After three washes, membranes were incubated with horseradish peroxidase‐conjugated goat antirabbit IgG antibody for 1.5 hr at room temperature. Then, washing was repeated four times, and the protein was visualized with an enhanced chemiluminescence kit (Sigma, USA). The density values of bands were quantified by densitometric analysis of scanned images (Scion Image 4.03). The relative protein ratio was calculated by determining the integrated intensity of the bands of each treated group as a ratio of the control condition.