(A) Immunoblots of in vitro ubiquitination reactions using human (hs) or mouse (mm) GSDMD and IpaH7.8. Results representative of 3 independent experiments.
(B) Immunoblots of 293T cells transfected with the GSDMD and IpaH7.8 constructs indicated. Results representative of 3 independent experiments.
(C and D) Immunoblots of 293T cells co-transfected with IpaH7.8 and GSDMD (wild-type, WT, or the mutants indicated). 3KR, K55R/K62R/K205R. 15KR, pan KR mutant. mm, mouse. hs, human. mm-K, introduces the 6 non-conserved lysine residues from human GSDMD into mouse GSDMD.
(E) Immunoblots showing co-immunoprecipitation of IpaH7.8(C357A) and GSDMD from co-transfected 293T cells.
(F) Binding isotherms of hsGSDMD (black circles) and mmGSDMD (turquoise circles) with IpaH7.8 measured by microscale thermophoresis (MST). The y-axis indicates the fraction bound, the x-axis is the Log10 of the IpaH7.8 concentration in molar. Each circle represents the mean ± SD of 3 technical replicates. Results representative of 3 independent experiments.
See also figure S3.