(A) Oligomerization assay to test the specificity of NAIP2.5 chimeras. HEK293T cells were transfected with NLRC4 and the indicated NAIP chimera and 6myc-tagged ligand. Cell lysates were subjected to blue native PAGE as in Figure 1. Results shown are representative of at least 3 independent trials. See also Figure S2.
(B) NAIP chimera responses to each ligand in (A) were quantified by densitometry of the oligomer species and normalized to wild-type NAIP2 or NAIP5 response to PrgJ or FlaA, respectively.
(C) Lysates from (A) were subjected to denaturing SDS-PAGE and immunoblotted for NLRC4, NAIP2, myc, and β-actin.
(D) NAIP5.2 inflammasomes are functional. HEK293T cells were transfected as in (A) but with the addition of CASP-1 and pro-IL-1β. Cell lysates were analyzed for IL-1β cleavage after 24 hours. Results shown are representative of 2 independent trials.