Figure 3

Strong activation of class Ia PI3 kinases by SCF, but PI3Kγ activation by Epo. Starved PEPs were mock-stimulated (m), stimulated with 0.3 U/ml Epo or with 25 ng/ml SCF with or without pretreatment with 100 nM WM as indicated. (A, B) The tyrosine-phosphorylated p85 regulatory subunits of activated class Ia PI3K enzymes (PI3Kα, β, δ) were immunoprecipitated from 500 μg total cell proteins with anti-phosphotyrosine mAb (4G10). To detect PI3K inositol kinase activity, immunoprecipitates were incubated with phosphatidylinositol (PI) and ³²P-γ-ATP. Phosphatidylinositol phosphate (PIP) generated by active PI3Ks was separated from ATP by thin layer chromatography (TLC) and analyzed by phosphoimaging. A representative example of the results from the phosphoimaged experiments is shown in (A) and the quantitative analysis of the results obtained with PEPs from three different cord blood samples in (B). Epo and SCF significantly activate class I PI3Ks (p _Epo < 0.01; p _SCF < 0.001) and WM significantly inhibits the SCF-induced activation (p < 0.01). (C, D) Starved PEPs were mock stimulated (m), stimulated with 0.3 U/ml Epo with or without pretreatment with 100 nM WM as indicated. PI3 kinase activity was detected as in (A) and (B) but using anti-p110γ to immunoprecipitate the catalytic subunit of PI3Kγ. A representative example of the results from the phosphoimaged experiments is shown in (C) (upper panel) and the quantitative analysis of the results obtained with PEPs from three different cord blood samples in (D). Significant PI3Kγ activation (p < 0.001) and inhibition by WM (p < 0.001) was determined. Equal PI3Kγ immunoprecipitation was confirmed by western blot (C, lower panel).