Anti gsdmd

The Western blot protocol used was previously described [7 (link)]. The primary antibodies included anti-TLR4 (Proteintech, China), anti-MyD88 (Proteintech), anti-NF-kB (R&D Systems, U.S.A.), anti-RIPK3 (Proteintech), anti-MLKL (Proteintech), anti-NLRP3 (ZEN BIO, China), anti-Caspase1 (Novus, U.S.A.), anti-Caspase8 (Proteintech), anti-GSDMD (ABclonal, China), anti-IL-1β (R&D Systems), and anti-β-actin (Elabscience, China). An anti–rabbit (or goat or mouse or rat) antibody (ABclonal) were applied to the secondary antibody.

Total protein of INS-1 cells was extracted and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) including the protease inhibitor (Roche, CA, USA). A BCA protein assay kit (Beyotime, Shanghai, China) was used to quantify proteins, and samples were mixed with 1 SDS sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromophenol blue). Proteins were separated in 12% SDS-PAGE, transferred onto the PVDF membrane, and immunoblotted with anti-GSDMD (1 : 1000, ABclonal, USA) and anti-NLRP3 (1 : 1000, ABclonal, USA) at 4°C overnight. Secondary antibodies (goat anti-rabbit IgG H&L (HRP), Abcam, USA) were applied for 2-3 h at room temperature, and membranes were developed via the BCA protein colorimetric assay kit (Beyotime, Shanghai, China). Developed protein bands were quantified by Tanon Gel Image System V2.0 program (Shanghai, China).

Cells were seeded on coverslips, and when the cells grew to 90%, they were infected with JEV at MOI = 1, and a blank control group was set up. Then, 48 h after infection, the cells were fixed with 4% paraformaldehyde, and the cells were permeabilized and sealed by conventional methods after fixation. Anti-GSDMD (ABclonal, Wuhan, China) and anti-JEV E (homemade) primary antibodies (1:200) were then incubated overnight at 4 °C. After washing, cells were incubated with FITC-conjugated and CY3-conjugated secondary antibodies (1:500) (ABclonal) for 1 h at room temperature. Finally, nuclei were stained with Hoechst and visualized with a fluorescence microscope.