2.4. Western blot analysis

Pulmonary tissue homogenate and MLE‐12 monolayer cultures were dissolved on ice with prepared RIPA buffer containing 1 mmol/L phosphatase inhibitor and 1 mmol/L PMSF for 30 minutes. The final lysate was collected, and the protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime). Equal amounts of protein were loaded for 10% SDS‐PAGE, and then, the proteins were transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk in TBS containing 0.1% Tween‐20 (pH 8.8) for 2 hours at room temperature. Then, the membranes were incubated with primary antibodies at 4°C overnight. The concentrations of all primary antibodies were as follows: anti‐p120, 1:5000 dilution (Abcam); anti‐phosphorylation NF‐κB and anti–NF‐κB, 1:1000 dilution (CST); anti‐NLRP3, 1:500 dilution (CST); and anti‐TLR4, 2 μg/mL dilution (Abcam). The membranes were washed with TBST and then incubated with appropriate horseradish peroxidase‐conjugated secondary antibody for 2 hours at room temperature. Finally, the protein bands were detected by chemiluminescence using an enhanced chemiluminescence (ECL) system. The density of all proteins was analysed using ImageJ software. Western blot bands were simply processed with Adobe Photoshop CS6.