Figure 6
Inhibition of Erks by PKC inhibitors but not by Raf inhibitor. (A, B) Starved PEPs were mock-stimulated (m) or stimulated with 0.3 U/ml Epo with or without pretreatment with 10 μM ZM336372 (ZM) for 1 h. B-Raf was immunoprecipitated from 500 μg total cell protein with anti-B-Raf. Precipitates were immunoblotted with anti-B-Raf 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 B-Raf activation from experiments with three different cord bloods is shown in (B) (p activation < 0.01; p inhibition < 0.05). (C) Starved PEPs were mock-stimulated (m) or pretreated with 0, 0.1, 1 or 10 μM ZM for 1 h or 4 h and stimulated with 0.3 U/ml Epo as indicated. Other cells were pretreated with 10 μM ZM and then stimulated with 25 ng/ml SCF. 100 μg total cell protein were immunoblotted with anti-P-STAT5, anti-P-MEK1/2, anti-Erk1/2 or anti-P-Erk1/2. Ø-lanes represent untreated PEPs. (D, E) Starved PEPs were mock-stimulated (m) or pretreated with PKC inhibitors and stimulated with 0.3 U/ml Epo or 25 ng/ml SCF. 100 μg of total cell protein were immunoblotted with anti-P-STAT5 and anti-P-Erk1/2. Inhibitor pretreatment was with 10, 100 or 1000 nM calphostin C for 1 h before Epo stimulation and with 1000 nM calphostin C before SCF stimulation. Other samples were pretreated with 0.1, 1, 10 or 100 μM Ro-31-8220 and stimulated with 0.3 U/ml Epo, or they were pretreated with 100 μM Ro-31-8220 where indicated and stimulated with 25 ng/ml SCF or 100 μM TPA for 10 min. 100 μg total cell protein were immunoblotted with anti-P-STAT5 or anti-P-Erk1/2. Ø-lanes represent untreated PEPs.