Anti gsdmd ab

Anti-mouse IL-1β Ab was purchased from Bioss (Beijing, China); Anti-caspase-1 (p20) mAb was purchased from AdipoGen (San Diego, CA); Anti-GSDMD Ab was purchased from Abcam (Cambridge, UK); Anti-JNK mAb and anti-phospho-JNK mAb were purchased from Cell Signaling Technology (Danvers, USA); Anti-β-actin Ab, HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Beyotime Biotech Co. Ltd (Shanghai, China). ELISA kits of mouse IL-1β, IL-6, IL-1α, IFN-γ and TNFα were purchased from Invitrogen (San Diego, CA).

Neutrophils were cultured with opti-MEM (Gibco, USA) in 12-well plates (5 ×10⁵ cells/well) for 12 h and then were stimulated with 1μg/mL SEO for 3 h. Next, 2 mM ATP (Sigma-Aldrich, St. Louis, MO) was added to the cells and incubated for an additional 9 h. After stimulation, the culture supernatants and cell lysates were collected as previously described (21 (link)). Protein concentrations were measured using Bradford protein Assay Kit (Beyotime, Beijing, China). Then proteins were subjected to 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. The membranes were blocked with 5% nonfat dry milk. Then, the membranes were immunoblotted with anti-IL-1β Ab (Bioss, Beijing, China), anti-Caspase1-p20 Ab (AdipoGen, USA), anti-GSDMD Ab (Abcam, Cambridge, UK), anti-JNK mAb (Cell signaling Technology, Danvers, USA), anti-phospho-JNK mAb (Cell Signaling Technology, Danvers, USA), streptavidin-horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) antibody, HRP-conjugated goat anti-mouse IgG (H+L) antibody (Beyotime, Beijing, China). β-actin was employed as a loading control for the cell lysates. Finally, the distinct protein bands were detected by ECL detection reagent (Beyotime, Beijing, China).