KL-induced activation of IL-7R in reconstituted 293T cells. (A) Tyrosine phosphorylation of γc and association of activated Kit with γc in response to KL. 293T cells were transiently transfected with constructs as indicated and stimulated with KL for 5 minutes. Immunoprecipitations of γc were analyzed with an anti-PY antibody detecting tyrosine-phosphorylated γc and γc-associated tyrosine-phosphorylated Kit (arrows, top panel, lane 2). The specificity of the protein association was verified by the control immunoprecipitation from KL-stimulated cells expressing Kit but lacking γc expression (lane 3). The blot was analyzed for the presence of γc protein (middle panel: short exposure; bottom panel: long exposure). (B) Tyrosine phosphorylation of IL-7Rα and association of IL-7R with activated Kit in response to KL. Analyses of IL-7Rα immunoprecipitates were performed as in panel A. (C) Independent tyrosine phosphorylation of γc and IL-7Rα by Kit. Kit was coexpressed with γc and IL-7Rα (lanes 1,2) or with γc or IL-7Rα alone (lanes 3 and 4, respectively). Immunoprecipitations against γc (top 2 panels) and IL-7Rα were performed and analyzed for tyrosine phosphorylation and presence of proteins as indicated.