Fig. 3 |. SpeB directly cleaves GSDMA after Gln246.

a, 293T cells transfected with the indicated plasmids were treated with or without the indicated inhibitors, before the whole cell lysates were analysed by western blot. SpeB is autocatalysed and the molecular weights of SpeB and its active autocatalysed fragment are approximately 42 kDa and 28 kDa, respectively. b, c, Coomassie blue-stained SDS–PAGE showing in vitro cleavage of recombinant GSDMA (1 μM) incubated with increasing concentrations of recombinant SpeB (12.5, 25, 50 and 100 nM, wedges) or mSpeB (250 nM) in the absence (b) or presence (c) of E64 (2 μM). WT SpeB was not detected, partially owing to the autocatalytic cleavage and reduced stability of active SpeB. d, Whole-cell lysates of 293T cells transfected with Flag-tagged gasdermins together with WT SpeB or mSpeB were immunoblotted with the indicated antibodies. e, Whole-cell lysates of A431 cells infected with GAS M1T1 strain 5448 (WT) or its isogenic mutant strains were analysed by western blot. f, Coomassie blue-stained SDS–PAGE showing in vitro cleavage of recombinant WT GSDMA or mutant GSDMA(I245N/Q246E) (1 μM) incubated with or without recombinant SpeB (100 nM). g, Whole-cell lysates of 293T cells transfected with the indicated plasmids were analysed by western blot. h, i, 293T cells stably expressing WT GSDMA or GSDMA(I245N/Q246E) were transfected by electroporation with recombinant SpeB or mSpeB and analysed by phase-contrast microscopy (h), for LDH release (i, top) and by western blot of whole-cell lysates with indicated antibodies (i, bottom). Cells exhibiting ballooning morphology (indicated by white arrows) are undergoing pyroptosis. Scale bar, 10 μm. Data in i are mean ± s.d. of triplicate wells. i, Two-tailed Student’s t-test. Data are representative of at least three independent experiments. CT, C terminal; FL, full-length; NT, N terminal. For gel source data, see Supplementary Fig. 1.