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. Author manuscript; available in PMC: 2022 Oct 13.
Published in final edited form as: Cell Host Microbe. 2021 Sep 6;29(10):1521–1530.e10. doi: 10.1016/j.chom.2021.08.010

Figure 3. IpaH7.8 targets human GSDMD, but not mouse GSDMD.

Figure 3.

(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.