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. 2017 Aug 10;8:231. doi: 10.1038/s41467-017-00235-x

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© The Author(s) 2017

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 — IL-7Rα glutamylation promotes Sall3 expression by STAT5. a–c Phosphorylation of STAT3 and STAT5 was tested by western blotting a, flow cytometry b and immunofluorescence staining c in CHILPs from WT and Ccp2 ^−/− mice after IL-7 stimulation. p-STAT5, green; p-STAT3, red; nucleus, blue. d Heat map of representative gene expression values from microarray data. In all, 1 × 10⁶ CHILPs (Lin⁻Flt3⁻CD25⁻IL-7Rα⁺α₄β₇ ⁺) from Ccp2 ^+/+ or Ccp2 ^−/− mice were sorted for microarray. e Analysis of indicated gene expression levels in Ccp2 ^+/+ and Ccp2 ^−/− CHILPs by quantitative reverse transcription PCR (RT-qPCR). Relative fold changes of gene expression values were normalized to endogenous Actb. f, g Enrichment assessment of STAT5 on Sall3 promoter in CHILPs from Ccp2 ^+/+ and Ccp2 ^−/− mice. h The association of STAT5 with Sall3 promoter was examined by EMSA. Sall3 promoter probe was biotin-labeled. i Flag-STAT5, pTK, and pGL3- Sall3 promoter were transfected into 293T cells for luciferase assay. j DNaseI accessibility of Sall3 promoter in CHILPs from Ccp2 ^+/+ and Ccp2 ^−/− mice was assessed. k H3K4me3 enrichment on Sall3 promoter was determined. CHILPs were isolated from Ccp2 ^+/+ and Ccp2 ^−/− mice, followed by ChIP assay. l Sall3 mRNA levels were detected in WT, Stat3 ^−/− _, and Stat5 ^−/− CHILPs. m CHILPs were isolated from WT mice and cultured with mitomycin C-treated OP9 cells for in vitro differentiation assay. Stat5 ^−/− CHILPs were transfected with STAT5 or Sall3 overexpression plasmid. Percentages of ILC3s were analyzed by flow cytometry. oe overexpression. n ILC3s were analyzed in Sall3 ^+/+ and Sall3 ^−/− mice. n = 6 for each group. o Histology of colons from Sall3 ^+/+ and Sall3 ^−/− mice 8 days after C. rodentium infection. Scale bars, 50 μm. p IL-7Rα glutamylation and STAT5 phosphorylation in CHILPs in the absence or presence of IL-7. CHILPs from WT or Il7r ^E446A BM were incubated with IL-7 and vehicle, followed by immunoblotting. β-actin was probed as loading controls. q Sall3 expression levels were tested in WT and Il7r ^E446A CHILPs by RT-qPCR. Relative fold change of Sall3 expression values were normalized to endogenous Actb. **P < 0.01, ***P < 0.001 (Student’s t-test). Data represent three independent experiments. Error bars in e–g, i–n, and q indicate s.d.