IL-4 does not phosphorylate Stat5 even in the absence of Stat6.

Splenic CD4⁺ T cells were purified from wild-type (WT) mice or Stat6^−/− mice and stimulated with platebound anti-CD3 mAb and anti-CD28 mAb for 48 hours. Cells were starved for 2 hours and stimulated with either IL-2 (5 ng/mL) or IL-4 (20 ng/mL) for 15 minutes. Shown is representative antiphospho-Stat5 (upper panel) and antipan Stat5 (lower panel) Western blotting from 4 independent experiments. Note that only a single band was detected with antiphospho-Stat5 antiserum, which was produced by immunization with a synthetic phosphopeptide corresponding to residues around Tyr694 of mouse Stat5a. The mobility of the band is identical to that of Stat5a but not of Stat5b.