IL-4 phosphorylates Stat6 in the absence of Stat5a.

(A,B) Expression of IL-4 receptor components on Stat5a^−/−CD4⁺ T cells. Freshly isolated splenic CD4⁺ T cells (A) or activated CD4⁺ T cells that were stimulated by anti-CD3 mAb and anti-CD28 mAb for 48 hours (B) from DO10⁺ mice or DO10⁺Stat5a^−/− mice were analyzed for the expression of IL-4Rα and γc by FACS. Shown are representative FACS profiles of IL-4Rα (left panels) and γc (right panels) staining from 4 independent experiments. Dashed lines are FACS profiles for the isotype-matched controls. (C) IL-4 induces Stat6 phosphorylation in the absence of Stat5a. Purified splenic CD4⁺ T cells from DO10⁺ mice or DO10⁺Stat5a^−/− mice were stimulated with platebound anti-CD3 mAb and anti-CD28 mAb for 48 hours. Cells were starved for 2 hours from cytokines and stimulated with IL-4 (20 ng/mL) for 15 minutes. Shown is representative antiphospho-Stat6 (upper panel) and anti-Stat6 (lower panel) Western blotting from 4 independent experiments. Stat6 was also equally phosphorylated in DO10⁺CD4⁺ T cells and DO10⁺Stat5a^−/−CD4⁺ T cells 30 minutes after IL-4 stimulation (data not shown).