AML blasts exhibiting constitutive Ser585 phosphorylation also show deregulated PI3-kinase signaling. Mononuclear cells from patients with AML (A, AML37; B-C, AML101) were stimulated with the indicated concentrations of GM-CSF for 5 minutes. Where indicated, cells were preincubated for 1 hour with 10μM H89 or DMSO vehicle control before GM-CSF stimulation. Cells were then lysed and βc immunoprecipitated and subjected to SDS-PAGE and immunoblot analysis with the indicated antibodies. (C) In vitro kinase assay for PI3-kinase activity was performed on βc immunoprecipitates with phosphatidyl inositol 4,5 phosphate (PIP) and ³²Pγ-ATP as described in “Cytokine signaling.” ³²P-PIP and the origin are indicated (C). Whole-cell lysates were blotted with anti–phospho-Ser473Akt (D) or anti–phospho-ERK (E), and signals were quantified by laser densitometry. The level of phosphorylation in the absence of GM-CSF was plotted as a percentage of maximum signal observed after GM-CSF stimulation. (F) Purified CD34⁺ cells from the indicated AML patient samples were treated with 10μM LY294002 or 0.1μM Wortmannin or DMSO for 24 to 72 hours in IMDM medium containing 0.5% FCS, after which time viable cell number was assessed with Flow-Count Fluorospheres, and total RNA was isolated for OPN quantitative RT-PCR as described in Figure 4D.