IL-21 does not inhibit IL-4–induced Stat6 phosphorylation or LPS-induced nuclear accumulation of NF-κB.

(A) Purified splenic B cells from BALB/c mice were incubated with IL-21 (10 ng/mL) for 2 minutes, 4 hours, or 12 hours before IL-4 stimulation and then stimulated with IL-4 (20 ng/mL) for 20 minutes. Whole cell lysates were prepared from the cells and subjected to Western blotting with antiphospho Stat6 antibody (top panels) or anti-Stat6 antibody (bottom panels). Shown are representative blottings from 4 independent experiments. (B) Purified splenic B cells were stimulated with IL-4 (20 ng/mL) in the presence or absence of IL-21 (10 ng/mL). At indicated times after IL-4 stimulation, whole cell lysates were prepared and subjected to Western blotting with antiphospho Stat6 antibody (top panels) or anti-Stat6 antibody (bottom panels). Shown are representative blottings from 3 independent experiments. (C) Purified splenic B cells were stimulated with IL-21 (10 ng/mL) or IL-4 (20 ng/mL) or both for 16 hours. Whole cell lysates were subjected to Western blotting with anti–Bcl-6 antibody. (D) Purified splenic B cells were stimulated with LPS (10 μg/mL) in the presence or absence of IL-21 (10 ng/mL) for 30 minutes. Nuclear lysates were prepared from these cells and the binding to a NF-κB consensus oligonucleotide was determined by electrophoretic mobility shift assays. In lane 5, a 50-fold molar excess of unlabeled competitor oligonucleotide (UO) relative to the labeled probe was added to the lysates.