ASC oligomerization assay

After inflammasome activation in the absence or presence of TCMD extracts, the cells were harvested, resuspended in cold lysis buffer containing Triton X-100 and complete protease inhibitor cocktail (Roche, Mannheim, Germany), and passed 10 times through a 27-gauge syringe. After centrifugation at 5000 rpm for 10 min, the supernatants (Triton-soluble fraction) were mixed with sample loading buffer (5×) and used to detect ASC (Cell Signaling Technology, Cat No. 67824) and β-actin (Santa Cruz Biotechnology, sc-47778). The remaining cell pellets were resuspended in PBS buffer containing 2 mM disuccinimidyl suberate (DSS, Sigma-Aldrich) cross-linker and were incubated at room temperature for 30 min, followed by centrifugation at 5000 rpm for 10 min. To detect ASC oligomerization, the cross-linked pellets (Triton-insoluble fraction) were separated on 12% SDS-PAGE and transferred to NC membranes. These membranes were probed with primary antibodies against ASC. After immunoblotting with HRP-conjugated goat anti-rabbit IgG (H+L) (Invitrogen) or goat anti-mouse IgG (H+L) secondary antibodies (Invitrogen), the proteins were detected using Clarity Western ECL Substrate (Bio-Rad).

Cells were harvested after inflammasome activation with or without TCMD extract, resuspended in 0.5 ml PBS buffer, and passed 10 times through a 27-gauge syringe. Cell lysates were centrifuged at 1000 rpm for 10 min to collect the cells.

Supernatants were diluted with equal volumes of lysis buffer containing triton X-100 and complete protease inhibitor cocktail (Roche, Mannheim, Germany), and centrifuged at 5000 rpm for another 10 min to pellet the ASC pyroptosomes. The pellets were resuspended in PBS buffer containing 2 mM disuccinimidyl suberate (DSS) cross-linker and were incubated at room temperature for 30 min followed by centrifugation at 5000 rpm for 10 min. The cross-linked pellets were fractionated on 12% SDS-PAGE, and ASC oligomerization was assessed by immunoblotting with ASC antibody (Cell signaling Technology; Cat No. 67824).