Fig. 5.

Vitamin B6 inhibits IL-33 stability through ubiquitin modification. a A549-IL-33 cells were cultured with or without 0.5 mM PL treatment for 24 h and then treated with CHX (20 µg/ml) for the indicated duration. The HA-IL-33/actin ratio is shown (n = 3). b, c A549-IL-33 cells were treated with PL at the indicated concentrations for 20 h and then with or without MG132 (10 μM) (a) or NH₄Cl (20 mM) (b). The intracellular IL-33 level was analyzed by western blotting. The HA-IL-33/actin ratio is shown (n = 3). d Determination of the effect of PL treatment on IL-33 ubiquitination. HEK293FT cells were transfected with the indicated plasmids and then treated with 0.5 mM PL (PL) for 24 h before being subjected to His pulldown. The data are representative of three independent experiments. e, f HEK293FT cells were transfected with full-length HA-IL-33 or mature IL-33 (112–270 aa) for 24 h and then treated with PL at the indicated concentrations. The western blot shows IL-33 expression. f Ratio of HA-IL-33 to actin expression (n = 2). g HEK293FT cells were transfected with full-length IL-33, mature IL-33 (112–270 aa) and Myc-Ub for 48 h and immunoprecipitated with an anti-HA antibody. The data are representative of three independent experiments. The data were analyzed by two-way ANOVA with Sidak’s multiple comparisons test (a–c) or one-way ANOVA with Dunnett’s multiple comparisons test (f). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant