Stat1, Stat3, and Stat5 are differentially activated by IL-21. (A) WT splenocytes were preactivated for 2 days with 2 ng/mL PMA + 1 μg/mL ionomycin, expanded with 10 U/mL IL-2 for 1 day, rested overnight in fresh medium without IL-2, and then stimulated with 100 U/mL IL-2 or 100 ng/mL IL-21 for 0 to 60 minutes. Cells were lysed and Western blotted with antibodies to phosphorylated Stat1 (p-Stat1, or p-S1), p-Stat3 (p-S3), or p-Stat5 (p-S5), and then reprobed with antibodies to Stat1 (S1), Stat3 (S3), Stat5a (S5a), or Stat5b (S5b). (B) Splenocytes were preactivated and rested as in panel A, and stimulated for 5 minutes with indicated concentration of IL-21 or IL-2. Cells were then harvested and Western blotted as in panel A. (C) CD8⁺ T cells from WT spleens (viability > 90%) were stimulated with IL-15 (100 ng/mL) or IL-21 (100 ng/mL) for 0 to 60 minutes, and lysates blotted as in panel A. We performed the Western blotting 3 times. In 2 experiments, the p-Stat1 signal in response to the combination of IL-15 and IL-21 was decreased, but in 1 it was increased. By flow cytometry for phosphorylated STAT proteins, which was performed 3 times, there was no difference. Thus, despite some experimental/technical variation, we do not believe that IL-15 + IL-21 cooperate to alter the level of p-Stat1 from what is seen with IL-21 alone.