Fig. 1: Phosphorylation of Smad3 linker Ser²¹³ site is essential for Th9 differentiation by TGF-β and IL-4 stimulation.

a Expression of Il9 mRNA in naïve CD4⁺ T cells from Tgfbr1^f/f ER-cre⁺ mice that had been treated with Oil (WT) or tamoxifen (Tgfbr1 KO) for 5 days stimulated with TGF-β plus IL-4 for 24 h. The results are presented relative to those of the control gene Hprt. b Western blot analysis of phosphorylated linker region Thr¹⁷⁹ (pSmad3L-T179), Ser²¹³ (pSmad3L-S213) sites and C-terminal (pSmad3C) of Smad3 and total Smad3 (Smad3) in WT and Tgfbr1 KO CD4⁺ T cells cultured with medium, TGF-β, IL-4 or TGF-β plus IL-4 for 2 h. c Western blotting of Smad3 phosphorylation in normal CD4⁺ T cells pre-treated with the indicated inhibitors and then cultured with TGF-β plus IL-4 for 2 h. d Real time RT-PCR analysis of Il9 mRNA in CD4⁺ T cells pre-treated with the indicated inhibitors as in c and then cultured with TGF-β plus IL-4 for 24 h. e IL-9 protein in cell culture supernatants were measured by ELISA in CD4⁺ T cells cultured with TGF-β plus IL-4 with or without p38 inhibitor for 72 h. f Phosphorylation of indicated Smad3 linker regions and C-terminal in Smad3^−/− CD4⁺ T cells transfected with intact WT Smad3, and S213A- or EPSM-Smad3 mutants (scheme depicted in Supplementary Fig. 2a–c), followed by TGF-β plus IL-4 stimulation for 2 h. g Intracellular staining of IL-9 by flow cytometry as in f after 72 h. h Summary of results in g. a, d These data were representative of four independent experiments or b, c, e–h pooled from three experiments. These data were analyzed by one-way ANOVA with Tukey’s test. Graphs show the mean ± SEM. Source data are provided as a Source Data file.