Fig. 1: Pre-BIR, BIR1, and HD1 domain of NAIP2 determine the specific binding to its ligand T3SS rod protein.

a Schematic domain of NAIPs predicted by the NCBI Conserved Domain Database. The NBD, HD1, WHD, and HD2 domains were annotated according to homology with NLRC4. The full-length NAIP protein is divided into fine fragments and illustrated by dash lines. Chimeras were constructed by exchange each fragments of NAIP2 with corresponding ones in NAIP5. The three critical amino acids that determines rod protein binding to NAIP2 are indicated by arrows on the top of the NAIP2 scheme, whereas below are sites for three different truncations. b Yeast two-hybrid assays of interactions between B. thailandensis rod protein BsaK and NAIP2/5 chimeras in which indicated NAIP2 region is substituted by NAIP5. c BsaK induced activation of the NAIP Chimera/NLRC4 inflammasome in reconstituted 293T cells. Lysates from 293T cells transfected with indicated plasmid combinations and stimulated with LFn-BsaK were analyzed for mature IL-1β (p17) by immunoblotting. d Yeast two-hybrid assays of interactions between BsaK and NAIP2 truncations indicated in a. e Blue Native PAGE assay to monitor the formation of the NAIP/NLRC4 inflammasome. 293T cells were transfected with NLRC4 and the indicated NAIPs or their truncation and 6-Myc tagged PrgJ or flagellin. After 36 h, cells were harvest and lysed in Native lysis buffer and subjected to Blue Native PAGE analysis. A fraction of the cell lysates were also subjected to denatured SDS–PAGE for protein expression and loading. f Yeast two-hybrid interaction assays of interactions between BsaK and different NAIP2 mutants. g Blue Native PAGE assay to test the ability of the NAIP2 mutants in forming inflammasome. h BsaK induced activation of the NAIP2 mutant/NLRC4 inflammasome in reconstituted 293T cells. Expression of transfected inflammasome components for c, e, g and h is in Supplementary Figure S1