Western Blotting Analysis

Cells were washed twice with ice-cold phosphate buffered saline (PBS) and then harvested and lysed in cold radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat milk and incubated with anti-NLRP3 antibody, ASC antibody, anti-caspase-1 antibody, and anti-IL-1β antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Blots were developed using enhanced chemiluminescent substrate (Thermo Fischer Scientific Pierce, IL, USA).