TCR stimulation, IL-2, and IL-15, but not IL-7, potently activate Rsk2 in mouse and human T cells. Mouse T cells were first activated by 2 μg/mL plate-bound anti-CD3 and 1 μg/mL anti-CD28 for 2 days and human PBLs were first activated by 2 μg/mL PHA for 3 days. The activated cells were expanded with 40 U/mL of IL-2 for 4 days (mouse) or 7 days (human), washed, and rested for 24 hours (mouse) or 48 hours (human) before restimulation with each reagent for 20 minutes as indicated. Total cell lysates were prepared and equal amounts of lysates were immunoprecipitated with anti-Rsk2 (C19 antibody; A,B) or anti-Stat5a and anti-Stat5b (C,D) antibodies, and phospho-proteins were detected by blotting using anti-pRsk (Thr359/Ser363; panel A lanes 1 through 5) for mouse pRsk2, anti-pRsk (Thr577; panel A lanes 6 through 11) for human pRsk2, or pY99 antibodies (C) for pStat5, respectively. Equal loading was confirmed by using anti-Rsk2 (E1; B) or pan-Stat5 (D) as indicated.