Disuccinimidyl suberate

For immunoblot of ASC oligomerization, BMDMs were lysed with Triton Buffer (pH 7.5 50 mM Tris HCl, 150 mM NaCl, 0.5% Triton X‐100, and 0.1 mM PMSF) for 10 min on ice. The cell lysates were centrifuged at 6,000×g for 15 min at 4°C and then resuspended in Triton Buffer and disuccinimidyl suberate (2 mM) (Sangon Biotech). After incubation for 30 min at 37°C for cross‐linking, cell pellets were washed by Triton Buffer for two times. Then cell lysates were centrifuged at 12,000×g for 15 min at 4°C, and were redissolved in 1× SDS loading buffer (Sangon Biotech) for immunoblot assays. For IF analysis of ASC speck formation, BMDMs washed with cold PBS three times, fixed in paraformaldehyde (4%, v/v) for 10 min, permeabilized with Triton X‐100 (0.1%, v/v) for 15 min, and blocked with BSA (3%, v/v). Cells were then stained with ASC antibody (1:200) overnight at 4°C and stained with Alexa fluor 488‐labeled secondary antibody (1:200) (Abcam) for 1 h at room temperature. Last, DAPI was used to stain cell nuclei. Cells were visualized using TCS SP2 confocal laser microscope (Leica).

After stimulation with nigericin (4 μM), BMDMs were washed with ice-cold PBS, and NP-40 lysis buffer (Beyotime, Shanghai, China) was then added to lyse cells for 30 minutes at 4°C. The samples were centrifuged at 350 × g for 10 minutes at 4°C. The pellets were rinsed with ice-cold PBS three times and resuspended in 500 μl of PBS. The pellet suspensions were crosslinked for 30 minutes with 2 mM disuccinimidyl suberate (Sangon Biotech, Shanghai, China) at room temperature. Next, the samples were centrifuged for 10 minutes at 350 × g at 4°C. The crosslinked pellets were mixed with 30 μl of sample buffer and then subjected to immunoblot analysis.