Anti gasdermin d

All western blotting procedures were performed as previously described (30 (link)) with the following primary antibodies: anti-PRX3, anti-NLRP3 (Abclonal Biotechnology, Wuhan, China), anti-gasdermin D (GSDMD), anti-caspase-1, anti-cleaved caspase-1, anti-IL-1β (all from Cell Signaling Technology, Boston, MA, USA), anti-IL-18, anti-PRX5 (Abcam, Cambridge, UK), PRX6 (Zen bio., Chengdu, China) and anti-β-actin (Bimake, Houston, TX, USA).

We loaded cell lysate containing ~15 µg protein into individual lanes, separated proteins using SDS-polyacrylamide gel electrophoresis, and then transferred proteins to a nitrocellulose membrane (0.22 µm pore size). We blocked membranes with 5% nonfat dry milk or bovine serum albumin (BSA) in PBS. We incubated membranes with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 h at room temperature. We used the following primary antibodies: anti-P65 (Cell Signaling Technology 8242), anti-phosphorylated P65 (Cell Signaling Technology 3033), anti-IL1B (ab9722 from Abcam, Cambridge, United Kingdom), anti-caspase 1 (Cell Signaling Technology 83383), anti-gasdermin D (Cell Signaling Technology 39754), anti-P62 (Progen GP62-C), and anti-beta actin (A5316 from Sigma, St. Louis, Missouri, USA). We visualized blots using the dual-channel Odyssey CLx Imaging System and quantified protein bands of interest using Image Studio. We normalized signal for proteins of interest against β-actin as a loading control.

Ultrapure LPS and nigericin were purchased from Invivogen and Sigma-Aldrich, respectively. The following antibodies were purchased commercially: rabbit anti-NLRP3 (cryo-2; Adipogen, AG-20B-0014), rabbit anti-ASC (AL177; Adipogen, AG-25B-0006), mouse anti-caspase-1 (p20) (Casper-1; Adipogen, AG-20B0042), mouse anti-caspase-1 (p10) (Casper-2; Adipogen, AG-20B0044), goat anti-mouse IL-1β antibody (R&D, AF-401-NA), anti-gasdermin D (Cell Signaling, #93709), anti-actin (clone 4; Merck Millipore, #MAB1501), anti-F4/80 (Cell Signaling, #70076).