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. 2023 May 23;20(7):794–807. doi: 10.1038/s41423-023-01029-6

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Fig. 7 — Vitamin B6 regulates IL-33 homeostasis by attenuating MDM2-mediated IL-33 ubiquitination. a HEK293FT cells were transfected with the indicated plasmids for 24 h, treated with or without 0.5 mM PL for 24 h, and immunoprecipitated with an HA antibody. Western blot analysis of IL-33 ubiquitination. b Western blot analysis of IL-33 ubiquitination in vitro. c, d Role of MDM2 in IL-33 ubiquitination. HEK293FT cells were transfected with the indicated plasmids and lysed before being immunoprecipitated with an anti-HA antibody. e Role of PL treatment in MDM2-mediated IL-33 ubiquitination. HEK293FT cells were transfected with the indicated plasmids for 24 h, treated with or without 0.5 mM PL for 24 h, lysed, and immunoprecipitated with an HA antibody. The V5-Ub/HA-IL-33 ratios in the immunoprecipitated groups are shown (n = 3). Two-way ANOVA with Sidak’s multiple comparisons test was used to analyze the data. ****p < 0.0001; ***p < 0.001; ns not significant. f Immunoblot analysis of lysates from HEK293FT cells transfected with the indicated plasmids and His-tagged MDM2 or the MDM2 RING domain truncation mutant and then treated as in (c). g HEK293FT cells were transfected with HA-IL-33 and increasing concentrations of full-length His-MDM2 and the truncated RING domain of His-MDM2 and analyzed by western blotting. The HA-IL-33/actin ratio is shown (n = 3). Ordinary one-way ANOVA with the Brown-Forsythe test was used to analyze the data. ****p < 0.0001; ns not significant. h Effect of the truncated 102–288 aa domain of MDM2 on IL-33 ubiquitination. HEL293FT cells were transfected with full-length MDM2 or truncated MDM2 and the indicated plasmids and immunoprecipitated with an HA antibody. i HEK293FT cells were transfected with HA-IL-33, and increasing concentrations of full-length His-MDM2 and the truncated 102–288 aa domain mutant of His-MDM2 were analyzed by western blotting. The HA-IL-33/actin ratio is shown (n = 3). One-way ANOVA with the Brown-Forsythe test was used to analyze the data. ****p < 0.0001; ns not significant. The results of one of three experiments are shown