2.6. Western Blotting

The protein samples obtained from cardiac tissues were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes. The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% skimmed milk and then incubated overnight with gentle shaking at 4°C in a diluent containing primary antibodies against NLRP3, IL-18, IL-1β, caspase-1, P-ERK, NF-κB, p-38, and anti-β-actin. The membranes were then incubated with a secondary antibody for 1 h. This analysis was performed independently three times. Protein levels were expressed as protein/β-actin ratios to minimize the loading differences. The relative signal intensity was quantified using the NIH ImageJ software.