Activation of Akt kinase in AML cells. (A) Cells from pheresis pack were thawed, washed, and immediately lysed. Lysates were analyzed for activated Akt kinase using an in vitro kinase assay as described in “Materials and methods.” CD34⁺ cells were purified as described and used as a control. Presence of phosphorylated GSK3 in the kinase reaction was detected by Western blotting using an anti–phospho-GSK3 antibody. To ensure that substrate was not lost in handling, blot was stripped and reprobed using an antibody that recognized total GSK3 (lower panel). (B) Patient samples were thawed, washed, and suspended in FACS buffer. Cells were stained with anti-CD45 antibody and analyzed for the presence of CD45⁺ side scatter intermediate cells. A representative dot plot is shown. Blast cells are within the gated population indicated by the circle. (C) Blast (B) and other cells (O) were sorted into separate populations. Cells were lysed immediately after sorting and Akt kinase assay performed as above. Samples from patient no. 134, which contains 98% blasts, were sorted in parallel and used as a control (lane 2).