Figure 4.

Stimulation of MAPK activity mediated by the EPO-R does not require maximal activation of the Shc/Grb2-Adapter pathway. After starvation, BaF3 cells expressing the wild-type EPO-R or the mutant EPO-Rs Y479F, F8, or F7Y479 were left unstimulated or stimulated with either 100 units EPO/ml (E) or 1 μg/ml interleukin 3. Lysates were subjected to immunoprecipitation with 5 μg polyclonal rabbit serum directed against Shc. Analysis of immunoprecipitated proteins was conducted by separation on a 15% low-bis SDS/PAGE and successive immunoblotting with anti-PTyr, anti-Shc, and anti-Grb2 antibodies. The positions of tyrosine-phosphorylated Shc (pY-Shc), Shc, and Grb2 are indicated by arrows.