Extended Data Fig. 5: Analyses of the binding interfaces between caspase-4 and pro-IL-18.
From: Recognition and maturation of IL-18 by caspase-4 noncanonical inflammasome
a, ITC profiles of the binding between caspase-4 (WT or an indicated mutant) and pro-IL-18 (WT or an indicated mutant). The C258A mutant was used for all caspase-4-p20/p10 in the assay. b, Calculated dissociation constant (KD) and binding stoichiometry (N) of ITC assays (a) are expressed as means ± s.d. from three determinations. ND, not detectable. c, e, Purified pro-IL-18 (WT or an indicated mutant) was incubated with caspase-4-p20/p10 (WT or an indicated mutant). Reaction mixtures were subjected to SDS-PAGE analyses. d, Approximate catalytic efficiency (kcat/Km) values of caspase-4 (WT or an indicate mutant), caspase-1, caspase-11, and caspase-5 in cleaving pro-IL-18. Purified pro-IL-18 was incubated with a titration series of the p20/p10 form of indicated caspases for 30 min. Reaction mixtures were subjected to SDS-PAGE analyses and quantification of the gel bands was performed to derive the kcat/Km. Calculated kcat/Km are expressed as means ± s.d. from three determinations. Mouse pro-IL-18 was assayed for caspase-11. f, Caspase-4 (WT or an indicated mutant) was co-expressed with pro-IL-18-Flag in CASP4−/−/GSDMD−/− (DKO) HeLa cells as in Fig. 3f. Cells were stimulated with LPS electroporation for 80 or 120 min, and cell lysates were analysed by immunoblotting. All data are representative of three independent experiments.
