Both MAPK and PI3K pathways contribute to IL-21–mediated proliferation. (A) Ba/F3 cells expressing WT IL-21R were starved of IL-3 for 5 hours and then stimulated with IL-21 (100 ng/mL) or IL-3 (2 ng/mL) for 5 minutes, 30 minutes, 1 hour, 3 hours, or 6 hours. Cells were harvested, lysed, and Western blotted with antibodies to p-Shc or p-Akt, and then reprobed with antibodies to Shc or Akt. Note that there are at least 3 isoforms for Shc (p46, p52, and p66). (B) Splenic CD8⁺ T cells were stimulated for 30 minutes with 100 ng/mL IL-15, 100 ng/mL IL-21, or both IL-15 and IL-21, harvested, lysed, and Western blotted as in panel A. (C) Ba/F3 cells stably transfected with the IL-21R-Y510 construct were stained with DDAO-SE, cultured in medium containing 10 ng/mL IL-21, 100 ng/mL IL-21, or 1 ng/mL IL-3 for 2 days in the presence of DMSO (control solvent), 50 nM wortmannin (Wort), 50 μM PD98059 (PD), or both Wort and PD, and analyzed by flow cytometry. Percent DDAO-SE dilution is indicated. (D) Splenic CD8⁺ T cells from Stat1^−/−Stat3^−/− mice were stained with CFSE, cultured in medium or with 100 ng/mL IL-15 and/or IL-21 for 4 days in the presence of DMSO, Wort, PD, or both Wort and PD, and analyzed by flow cytometry. Percent CFSE dilution is indicated. Results are representative of 3 experiments. Statistical analyses are comparisons to cells treated with DMSO. *P < .05; **P < .01; ***P < .001.