Anti caspase 1

For liver specimens, liver sections were permeabilized with 0.5 % Triton X-100 for 20 min and blocked in 5 % BSA solution for 40 min in room temperature, followed by incubating with primary anti-Caspase-1 (Affinity Biosciences, Zhenjiang, China), anti-NLRP3 (Abcam, Cambridge, UK) and anti-IL-1β (Abcam, Cambridge, UK) antibodies overnight at 4°C. Then, liver sections were stained with fluorescent secondary antibodies conjugated with Alexa Fluor 594 (ThermoFisher, Madrid, Spain). For nuclear counterstaining, 4,6-diamidino-2-phenylindole (DAPI, KeyGEN, Nanjing, China) was applied, and cells were observed using a fluorescence microscope (Nikon).

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.)