BMDM cells were permeabilized with TBS buffer (50 mM Tris-HCl and 150 mM NaCl) with 0.5% Triton X-100 with protease inhibitors (Thermo Fisher Scientific) and lysed by passing through a 21-gauge needle, then centrifuged for 10 min at 4 °C. The pellets and the supernatants were used as Triton-insoluble and soluble fractions. The Triton-insoluble fractions were further resuspended in TBS buffer, then cross-linked by treatment with 2 mM disuccinimidyl suberate (DSS) (Thermo Fisher Scientific) at room temperature for 30 min. The lysates were analysed by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), followed by western blotting assays with rabbit anti-ASC antibody.
