Rsk1, Rsk2, and Rsk3 are activated in human T cells by IL-2 and IL-15 but not by IL-7. (A) Total lysates from YT cells not treated or treated with IL-2 for various time points were subjected to an on-membrane in vitro kinase assay. The arrow indicates the IL-2–induced protein kinase activity. (B) Total lysates from YT cells or primary human T cells not treated or treated with IL-2 for 15 minutes were immunoprecipitated with anti-Rsk1, anti-Rsk2, or anti-Rsk3 antibodies and the corresponding immune complexes then subjected to an on-membrane in vitro kinase assay. The arrow indicates the protein kinase activity. Levels of immunoprecipitated Rsk1, Rsk2, and Rsk3 were determined by blotting an aliquot with antibodies as indicated. Vertical lines have been inserted to indicate repositioned gel lanes. (C) Total lysates from primary human T cells not treated or treated with IL-15 or IL-7 for 20 minutes were immunoprecipitated with anti-Rsk1, anti-Rsk2, anti-Rsk3, or anti-Stat5a/b antibodies. Phospho-Rsk1 and -Rsk2 were detected by blotting with anti-pRsk (Thr577), phospho-Rsk3 with anti-pRsk (Thr359/Ser363), and phospho-Stat5 with pY99 as indicated. Equal amounts of protein immunoprecipitated by each antibody were confirmed by blotting with each corresponding antibody as shown.