Sds loading buffer

For immunoblotting analysis, whole-cell extracts were lysed with low-salt lysis buffer, and supernatants were precipitated with methanol/chloroform, boiled for 5 min with SDS loading buffer (Cell Signaling Technology, Danvers, MA, USA) and resolved on SDS-PAGE gels. The proteins were transferred to PVDF membranes (BioRad) and further incubated with the indicated antibodies. LumiGlo Chemiluminescent Substrate System from KPL (Gaithersburg, MD) was used for protein detection. Images were cropped for presentation and the full size images are presented in Supplementary Fig. 8.

Cells were lysed by low-salt lysis buffer. For endogenous IP, whole-cell lysates were treated with indicated antibodies overnight and then incubated with protein A and G beads (Pierce) for 4 to 6 hours. For exogenous IP, whole-cell lysates were only incubated with anti-FLAG or anti-MYC (EQKLISEEDL) agarose gels. Immunoprecipitates were eluted with 2× SDS loading buffer after washing five times with low-salt lysis buffer. Cells were lysed by low-salt lysis buffer, boiled for 5 min with SDS loading buffer (Cell Signaling Technology), and resolved on SDS–polyacrylamide gel electrophoresis gels. Proteins were transferred to a polyvinylidene difluoride membrane (MilliporeSigma). The membranes were blocked with 5% (w/v) reagent-grade nonfat milk and further incubated with the antibodies as listed in the “Antibodies” section. EMD Millipore Luminata Western HRP Chemiluminescence Substrate was used for protein detection for all blots.

THP-1-derived macrophages or BMDMs were seeded in 6-well plates and treated as indicated. The cell pellets were collected into 1.5 ml Eppendorf tubes and lysed in TBS buffer (50 mM Tris-HCl (GCRF, China), 150 mM NaCl (GCRF, China), 0.5% Triton X-100 (Sigma-Aldrich), PH 7.4) with phosphatase inhibitor (Roche) and EDTA-free protease inhibitor (Bimake) on a rocker for 30 min on ice, and then centrifuged at 6000 × g/4 °C for 15 min to discard the supernatants. The cell supernatants were harvested for precipitation and detected the activation of caspase-1 (p10) and the maturation of IL-1β (p17) by western blotting. The pellets were washed twice with TBS buffer and resuspend in TBS buffer containing 2 mM fresh disuccinimidyl suberate (DSS, Thermo Fisher Scientific) cross-linker at 37 °C for 30 min to crosslink with flipping the tubes every 10 min, and then spun at 6000 × g/4 °C for 15 min. The crosslinked pellets were resuspended in 25 μl 2× SDS loading buffer (Cell Signaling Technology) and boiled at 100 °C for 5 min, and analyzed by immunoblotting of anti-ASC antibody, anti-BAX antibody or anti-BAK antibody.

For immunoprecipitation, whole-cell extracts were acquired after transfection or stimulation with appropriate ligands, followed by incubation with anti-myc beads or the appropriate antibodies plus Protein A/G (Pierce). Beads were then washed five times with low-salt lysis buffer, and immunoprecipitates were eluted with 2× SDS Loading Buffer (Cell Signaling Technology) and resolved by SDS–polyacrylamide gels, and then transferred to NC membranes. Primary antibodies against indicate genes and anti-myc beads were used according to manufacturer’s instructions. Peroxidase conjugated secondary antibody (CST) was used, and the antigen-antibody reaction was visualized by enhanced chemiluminescence assay (ECL, Thermo). The antibodies used are listed in Supplementary Table 9.