ASC oligomerization assay

THP-1 cells were lysed in buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, and 320 mM sucrose) supplemented with a protease inhibitor cocktail. The nuclei and unlysed cells were removed by centrifugation and 5 μm Ultrafree-CL centrifugal filters (Millipore). The filtrate was diluted with an equal volume of CHAPS buffer (20 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, and 0.1% CHAPS) supplemented with a protease inhibitor cocktail, and centrifuged to obtain insoluble pelleted fraction. The pellets were resuspended in CHAPS buffer and then subjected to cross-linking using disuccinimidyl suberate (DSS; 2 mM) for 30 min. The protein samples were resolved by 12% SDS-PAGE, and the level of ASC was analyzed. For the reconstitution of NLRP3 inflammasomes, HEK293T cells were transfected with pCMV-Flag-NLRP3, pLKO_AS2-ASC-Flag, and pLKO_AS2-ASC (Y146F)-Flag using Lipofectamine 2000. They were then incubated for 48 h and analyzed as described for THP-1 cells. ASC speck formation was analyzed in ASC-mCherry-expressing THP-1 cells. ASC speck images were acquired under fluorescence microscopy (Olympus). For quantification, the ASC speck was counted with an IN Cell Analyzer (GE Healthcare) and normalized with respect to the number of nuclei, which were stained with DAPI.