Structurally and mechanistically distinct PI3 kinase inhibitors prevent Epo-induced activation of Ras, MEKs and Erks. (A) Starved (st) PEPs were mock-stimulated (m) or pretreated with 0, 25, 50 or 100 nM wortmannin (WM) for 30 min or with 30 or 100 μM LY294002 (LY) for 1 h and then stimulated with 0.3 U/ml Epo where indicated. For comparison, PEPs starved and pretreated with 100 nM WM or 100 μM LY where indicated were stimulated with 25 ng/ml stem cell factor (SCF) for 10 min to activate c-Kit signaling. 100 μg total cell protein were immunoblotted with P-STAT5, P-Akt or P-Erk1/2 antibodies as indicated. GTP-loaded Ras was precipitated with GST-c-Raf1 RBD from 500 μg total cell protein and immunoblotted with anti-Ras. Ø indicates non-starved and non-treated PEPs. (B) PEPs pretreated with 100 nM WM for 30 min where indicated were mock-stimulated (m) or treated with 0.3 U/ml Epo or 25 ng/ml SCF for 10 min. 100 μg total cell protein were immunoblotted with P-MEK1/2, Erk1/2, P-Erk1/2 or P-GSK3α/β antibodies as indicated. Phosphorylated EpoR was immunoprecipitated with anti-phosphotyrosine mAb (4G10) from 500 μg cell protein and immunoblotted with anti-EpoR. Ø indicates non-starved and non-treated PEPs.