TSLP-induced STAT5 phosphorylation requires the presence of at least one cytoplasmic tyrosine residue. Exponentially growing Ba/F3 cells expressing the indicated receptors were washed three times with RPMI 1640, deprived of IL-3, and then left untreated or stimulated with recombinant human TSLP or mouse IL-3 as shown. After cell lysis, the cell lysates were subjected to immunoprecipitation using anti-STAT5a antibodies. After washing, the immunoprecipitates were resolved by SDS-PAGE. The phosphorylation status of STAT5a was assayed by Western blotting with anti-phosphotyrosine antibodies (4G10) and the total amount of Stat5a was measured by reprobing with anti-Stat5a antibodies as indicated.