Anti cleaved caspase 1

The pre-cooled PBS was used to rinse the cells in 24-well plates (2 × 10⁴ cells/well) for 3 times and 4% paraformaldehyde was employed to fix the cells deposited in plates of 24-well. After that, cells were subjected to permeabilization with PBS containing 0.1% Triton X-100 for 10 min at 37 °C. Then, 3% BSA was added for blockading the nonspecific binding. Next, the cells were incubated with the anti-VP1 (1:1000 dilution; Millipore, USA), anti-cleaved-Caspase1 (1:100 dilution; Affinity, USA) and anti-NLRP3 antibody (1:100 dilution; Affinity, USA) overnight at 4 °C, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG and Daylight 594-conjugated donkey anti-rabbit IgG secondary antibodies (1:300 dilution; CST, USA) for 1 h. Finally, PBS was adopted to rinse the cells for 3 times and diamidino-2-phenylindole (DAPI; 1:1000 dilution; Beyotime, China) was taken to stain them at 37 °C for 5 min. With the help of a confocal fluorescence microscope (Leica, Germany), the images were obtained.

A total of 1×10⁶−10⁷ cells were collected and lysed using RIPA lysis buffer on ice for 15–30 min and centrifuged at 4°C, at 12,000-14,000 × g for 10 min. The supernatant was collected, 1× Loading buffer was added, and the samples were denatured by heating in a metal bath at 100°C for 10 min and stored at −20°C for later use. Equal quantities of protein were loaded on a 12% SDS-gel (YESEN, China), resolved using SDS-PAGE, transferred to methanol-activated PVDF membranes, blocked with 5% skimmed milk for 15–30 min, and incubated with different primary antibodies at 4°C overnight. The following day, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 45–60 min at 25°C. Signals were visualized using an Immobilon Western Kit (MilliporeSigma). The antibodies used in this study were: Anti-GSDMD (1:2,000; cat. no. AF4012; Affinity Biosciences); Anti-Cleaved-Caspase-1 (1:2,000; cat. no. AF4005, Affinity Biosciences); anti-Caspase-1 (1:1,000; cat. no. ab138483; Abcam); anti-β-actin [1:2,000; cat. no. 30102ES60; Yeason Biotechnology (Shanghai) Co., Ltd.], and anti-rabbit mAb (1:4,000, cat. no. 5571S; Cell Signaling Technology, Inc.)