NLRP1 T755N is GOF for inflammasome activation. (A) Western blot showing similar expression of NLRP1 WT and T755N protein in HEK293T cells. A GAPDH Western blot is shown as a loading control. The image is representative of 3 independent experiments. (B) Western blot for HA-tagged NLRP1 after BN-PAGE or conventional SDS-PAGE and lysates from HEK293T cells overexpressing cDNA of NLRP1 WT (2 replicates), T755N, previously published GOF alleles (M77T and F878_R843del), or noncleavable NLRP1 T755N (T755N+F1212A), demonstrating oligomerization of T755N NLRP1 similar to other GOF mutations. GAPDH Western blot is shown as a loading control. (C) ELISA of IL-1β in supernatants of keratinocytes after transfection with NLRP1 alleles demonstrating that T755N is GOF for IL-1β production and that cleavage at the F1212 position is required for IL-1β production. NT, nontransfected cells; EV, empty vector. Data are an average of 4 replicates. ***P < 0.001, 1-way ANOVA.