Figure 3.

Stimulation of MAPK activity mediated by the EPO-R involves recruitment of PI3 kinase but does not require maximal activation of PKCα. BaF3 cells expressing the wild-type EPO-R or the mutant EPO-R F7Y479 were either left untreated or were pretreated with TPA as described in A, indicated by + TPA (18 h). Prior to lysis cells were left unstimulated (−) or were stimulated either for 5 min with 100 units EPO/ml (+) or for 10 min with 1 μg/ml TPA (+). MAPK activity was immunoprecipitated using an antiserum directed against ERK2 (IP: αERK2) and subjected to an in vitro kinase assay in the presence of MBP and [γ-³²P]ATP as described. MBP-phosphorylation was detected by analysis on a 15% SDS/PAGE, transfer to a nitrocellulose membrane, and autoradiography (Upper). Immunoblotting with an antiserum against ERK2 (IB: αERK2) demonstrates equal loading in all lanes (Lower).