Inflammasome Activation Assay: Unlocking Immune Responses

The inflammasome assay was performed as previously described with some modifications^{3 (link),4 (link)}, all experiments were repeated three times. Briefly, plasmids of pCMV-mPro-IL-1β (100 ng, Addgene no. 73953, a gift from C. Stehlik⁴⁴ ), pCMV-mCaspase1-C-flag (10 ng, Addgene no. 21142, a gift from J. Yuan⁴⁵ ), pCS2-Myc-NLRC4 (300 ng, a gift from F. Shao), pcDNA3.4-Flag-NAIP5 WT/mutant (300 ng) were cotransfected into HEK293T cells (gifted from X. Xiao) in a 12-well plate. After 24 h of transfection, LFn-FlaA and protective antigen proteins were added to the culture medium to a final concentration of 10 μg ml⁻¹. The same concentrations of LFn-FlaAAA and protective antigen were added to the negative control well. After another 24 h, the transfected cells were gathered in a passive lysis buffer (250 μl per well; Promega no. E194A) and obtained supernatants were subjected to western blot analysis. The detection of cleaved IL-1β (p17) on an immunoblot confirmed the occurrence of active inflammasome complex formation. Antibodies used were: anti-IL-1 (Abcam no. 234437), anti-Flag (Sigma no. F1804), anti-Myc (Cell Signaling no. 2276), anti-Caspase-1 (Cell Signaling no. 24232), antimouse IgG (Cell Signaling no. 7076), antirabbit IgG (Cell Signaling no. 7074S) and anti-β-tubulin (Invitrogen no. MA5–16308-HRP).