A functional hIL-5R complex is formed only after IL-5 stimulation, followed by JAK2 and JAK1 activation. (A) Unstimulated, IL-5–stimulated, mono5-stimulated, or GM-CSF–stimulated TF-h5Rα cells were lysed and immunoprecipitated with KM1266 (anti–hIL-5Rα MoAb) or anti-βc MoAb. The immunoprecipitated proteins were analyzed on SDS-PAGE and transferred to Immobilon membranes. The membranes were probed with anti-βc polyclonal Ab or KM1074 (anti–hIL-5Rα MoAb), respectively. The same membranes were reprobed with anti–hIL-5Rα or KM1074. Another immunoprecipitation analysis was made by immunoblotting with antiphosphotyrosine MoAb. (B) Unstimulated, IL-5–stimulated, or GM-CSF–stimulated TF-h5Rα cells were lysed, and immunoprecipitation was performed with anti-JAK1 or -JAK2 Ab. The immunoprecipitates were analyzed on SDS-PAGE and transferred to Immobilon membranes. The membranes were probed with antiphosphotyrosine MoAb 4G10 and reprobed with anti-JAK2 or -JAK1 Ab.