Time course of JAK phosphorylation and JAK/c-Mpl coimmunoprecipitation. Ba/F3-mMPL cells were stimulated with TPO (2% conditioned medium) for 0 to 60 minutes, and cellular lysates were obtained. Immunoprecipitations were performed with the indicated antisera and were analyzed by Western blot. (A) Phosphotyrosine immunoblots (P-Tyr) show the kinetics of MPL, JAK2, and TYK2 phosphorylation in response to TPO. (B) Samples were immunoprecipitated with MPL-specific antiserum and probed to detect coimmunoprecipitation of JAK2 and TYK2 (arrows indicate the position of each protein). The blots were stripped and reprobed to confirm equal immunoprecipitation and loading (not shown).