Figure 5
Activation of B-Raf by Epo is blocked by wortmannin. PEPs were mock-stimulated (m) or stimulated with 0.3 U/ml Epo or pretreated with 100 nM WM where indicated and then stimulated with Epo. (A, B) c-Raf1 was immunoprecipitated from 500 μg total cell protein with anti-c-Raf1. Precipitates were immunoblotted with anti-c-Raf1 for IP-control (A, lower panel) or incubated with GST-MEK and subsequently GST-ErkK63M and 32P-γ-ATP for coupled kinase assay. Proteins were separated by SDS-PAGE and phosphorylated GST-Erk1K63M analyzed by phosphoimaging. A representative example is shown in (A) (upper panel). Quantification of c-Raf1 activation from experiments with three different cord bloods is shown in B (p activation < 0.001; p inhibition < 0.05). (C, D) B-Raf activation was analyzed as described in (A) and (B) but anti-B-Raf was used for immunoprecipitation and immunoblotting. Both activation and inhibition were statistically highly significant (p activation < 0.001; p inhibition < 0.01).