Partial reconstitution of Kit^YY567/569FF through transactivation of IL-7R requires tyrosine residues of IL-7R. (A) IL-7–mediated activation of Stat5 is dependent on tyrosine residues within IL-7R. Lysates of 293T cells coexpressing Jak3 and IL-7R^WT (lanes 1,2) or a mutant IL-7R lacking all cytoplasmic tyrosine residues (γc^Y/F and IL-7Rα^Y/F, lanes 3,4) were analyzed for phosphorylation of Stat5 on Tyr⁶⁹⁴ (top panel) and Stat5 protein (bottom panel). (B) Requirement for cytoplasmic tyrosines of IL-7R for Kit^YY567/569FF-induced Stat5 activation. Lysates from 293T cells coexpressing Kit^YY567/569FF and IL-7R^WT (lanes 1,2) or the mutant IL-7R consisting of γc^Y/F and IL-7Rα^Y/F (lanes 3,4) were analyzed for phosphorylation of Stat5 on Tyr⁶⁹⁴ Stat5, Kit, γc, and IL-7Rα protein levels. (C) Quantification of 4 independent experiments analyzing Kit^YY567/569FF-induced (left) or IL-7R-induced (right graph) phosphorylation of Stat5 in presence (wt) or absence (Y/F) of cytoplasmic tyrosine residues of IL-7R. Stat5 phosphorylation was determined by analyzing either lysates or immunoprecipitates of Stat5 for phosphorylation of Stat5 and protein levels of Stat5. Data are displayed and statistically analyzed as in Figure 4C.