Immunoblot Analysis of Protein Expression

For immunoblot analyses, modified RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) was used to extract total protein from frozen lung tissue [24 (link)]. Extraction proteins from tissues and cells were measured using the BCA protein assay reagent kit (Pierce). An equal amount of total protein (80 μg of protein/lane) was resolved on a 5–12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat dry milk in PBST (containing 0.05% Tween 20). Incubation with the antibodies was performed using the following dilutions: 1:750 for P2X₇R (Abcam, USA) and 1:1000 for caspase-1, procaspase-1, caspase-1, IL-1β (all of them from Cell Signaling Technology, USA) and total NLRP3 (Biosource, Belgium). Primary antibodies were detected with horseradish peroxidase-conjugated antibodies, 1:5000 for anti-mouse (ZSJQ-BIO, Beijing, China) and 1:5000 for anti-rabbit (ZSJQ-BIO, Beijing, China), at room temperature for 1.5 h. Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualised using a FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA). Blot bands were qualified using NIH ImageJ software.