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. 2023 Jan 4;14:61. doi: 10.1038/s41467-022-35725-0

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Quantification of the binding affinity between GSDMB and IpaH7.8 by ITC. b The mutations on IpaH7.8 impair the binding of GSDMB. The protein complex was immunoprecipitated with anti-FLAG antibody, and analyzed by western blotting with the indicated antibodies. c Ubiquitination assay for GSDMB by IpaH7.8. Mutations on IpaH7.8 failed to efficiently ubiquitinate GSDMB. Ub, anti-ubiquitin antibodies. d In vitro pull-down of IpaH7.8 by MBP-tagged WT or mutant GSDMB proteins. e The co-immunoprecipitation assay of IpaH7.8 with WT or mutant GSDMB proteins. f Disruption of the GSDMB-IpaH7.8 interactions compromises the ubiquitination of GSDMB. Ub, anti-ubiquitin antibodies. g NK cell-mediated killing of intracellular WT or Δipah7.8 S. flexneri. The colony-forming units (CFU) of S. flexneri recovered from HEK293T cells bearing GSDMB D17A or I97D mutant were significantly reduced when co-cultured with NK cells. All experiments were repeated at least three times. Statistical differences were calculated using Two-way ANOVA (mean ± sd, n = 3 independent biological replicates, ns (no significant): p = 0.4508, ****p < 0.0001). Source data are provided as a Source Data file.