IL-21 does not inhibit Stat6-mediated transcription from germ line Cε promoter construct.

(A) IL-21 induces phosphorylation of Stat1, Stat3, and Stat5 in B cells. Purified splenic B cells, M12.4.5 cells, and A20 cells were stimulated with IL-21 (10 ng/mL) for 20 minutes. Ba/F3 cells were starved from IL-3 for 2 hours and then stimulated with IL-21 (10 ng/mL) for 20 minutes. Whole cell lysates were prepared and subjected to Western blotting with antiphospho Stat1 antibody, antiphospho Stat3 antibody, or antiphospho Stat5 antibody. Positive controls (PC) were lysates of IFN-γ–stimulated splenocytes, IL-6–stimulated splenocytes, and IL-7–stimulated thymocytes for antiphospho Stat1 blotting, antiphospho Stat3 blotting, and antiphospho Stat5 blotting, respectively. Negative controls (NC) were unstimulated splenocytes and unstimulated thymocytes for Stat1 and Stat3 and for Stat5, respectively. Shown are representative blottings from 3 independent experiments. (B-D) IL-21 does not inhibit the transcription from Stat6-dependent reporter plasmids. M12.4.5 cells were transfected with either ε-162Luc (B), ε-623Luc (C), or TPU474 (D) in the presence of murine Stat6 expression plasmid (pcDNA3 Stat6) and pRL-TK. Twelve hours after transfection, cells were stimulated with IL-4 (20 ng/mL) or IL-21 (10 ng/mL) or both for another 12 hours and the luciferase activity was measured by the dual luciferase reporter system. Data are means ± SD from 4 experiments.