ASC Oligomerization Assay

Macrophages were harvested in lysis buffer (50 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl, 10% glycerol, 0.5% Triton X‐100, 1 mmol/L PMSF, and complete protease inhibitor cocktail) and incubated on ice for 30 minutes, followed by centrifugation at 6000g for 15 minutes at 4°C. The supernatants and pellets were used as the Triton‐soluble and ‐insoluble fractions, respectively. For detection of ASC oligomerization, the Triton‐insoluble fractions were washed with lysis buffer and the pellets were resuspended in 300 μL of lysis buffer. The pellets were crosslinked for 30 minutes at 37°C with 2 mmol/L disuccinimidyl suberate (Pierce) and then spun down for 15 minutes at 6000g. The pellets were eluted and analyzed by immunoblotting with anti‐ASC.