(A) Tyrosine phosphorylation of Epo-R. Factor-deprived E4 cells were incubated with medium (control), 500 U/mL IL-6 (lane IL-6), or 10 U/mL Epo [lanes Epo and Epo(D)] for 5 minutes. Cell lysates were prepared as described in the Materials and Methods and proteins immunoprecipitated with anti–Epo-R antibodies once (Epo) or twice [Epo(D)] were separated by SDS-PAGE under reducing conditions and blotted. The filter was probed with an antiphosphotyrosine antibody (4G10). (B) Tyrosine phosphorylation of Jak2. Factor-deprived E4 cells were incubated with medium (control), 500 U/mL IL-6 (lane IL-6), or 10 U/mL Epo (lane Epo). After lysis, proteins were immunoprecipitated with anti-Jak2 antibodies, separated by SDS-PAGE under reducing conditions, and blotted. The filter was probed with an antiphosphotyrosine antibody (4G10).