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. 2014 Dec 15;211(13):2651–2668. doi: 10.1084/jem.20132681

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© 2014 Gao et al.

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Figure 6. — IL-2-STAT5 signaling and NF-κB activation in E-protein KO mice. (a) CD4⁺ cells were purified from WT and floxed E2A/HEB ER-Cre mice and were cultured in the presence of TGF-β and 2 µM 4-OHT for 3 d. Foxp3 (GFP)⁺ cell and CD25 expression was evaluated by flow cytometry. Data are representative of three independent experiments. (b) CD4⁺ cells purified from WT Foxp3KI and floxed E2A/HEB Er-Cre/Foxp3-GFP-KI mice cultured as in (a) with or without IL-2. Foxp3 (GFP)⁺ cells and CD25 expression were evaluated by flow cytometry; histogram on the right shows CD25 expression. Data are representative of two independent experiments. (c) CD4⁺ cells were purified from WT and floxed E2AHEB ER-Cre mice and cultured as described in (a), and pSTAT5 was evaluated by flow cytometric analysis. Data are representative of three independent experiments. (d) CD4⁺ cells were purified from WT and floxed E2AHEB ER-Cre mice and cultured under Th0 (no cytokine) or iT reg cell conditions in the presence of 2 µM 4-OHT for 3 d; Expression of pSTAT5, STAT5, pSmad3, and Smad3 was assessed by immunoblot. Data are representative of three independent experiments. (e) Frequency of CD25 (IL-2Rα)⁺/CD122 (IL-2β)⁺ cells among CD4SP thymocytes from tamoxifen-treated WT and floxed E2A/HEB ER-Cre mice was assessed by flow cytometry. Numbers in quadrants indicate percentage. Data shown are representative at least three independent experiments. (f) STAT5 phosphorylation in CD4SP and DP thymocytes from tamoxifen-treated WT and floxed E2A/HEB ER-Cre mice was assessed by flow cytometry. Data shown are representative of three independent experiments (g) Whole-cell extracts were prepared from thymocytes of tamoxifen-treated WT and E2Af/fHEBf/f ER-Cre (KO) mice, and expression of pSTAT5, STAT5, E47 and actin was assessed by immunoblot. Data shown are representative of three independent experiments. (h) CD4⁺ cells were purified from WT and floxed E2AHEB ER-Cre mice and cultured under Th0 (no cytokine) or iT reg cell conditions in the presence of 2 µM 4-OHT for 3 d. Expression of c-Rel, p50, p65, HEB, and actin was assessed by immunoblot. Data shown are representative three independent experiments. (i) Whole-cell extracts prepared as in (g). c-Rel, p50, p65, E2A, IκBα, and actin were assessed by immunoblot. Data shown are representative of three independent experiments. (j) Whole-cell extracts prepared as in (g). p-IκBα, IκBα, and actin were assessed by immunoblot. Data shown are representative of three independent experiments. (k) Whole-cell extracts prepared as in (g). Iκκβ, IκBα and actin were assessed by immunoblot. Data shown are representative of three independent experiments.