Akt phosphorylation in AML cells. (A) Patient samples were thawed, washed, harvested by centrifugation, and lysed in 1% Triton-X 100 lysis buffer; 100 μg cell lysates were loaded on SDS-PAGE gel and analyzed by Western blotting. Blots were probed with phospho-Akt antibody (top panel) and then stripped and reprobed with total Akt antibody (middle panel). As a loading control, lower portion of blot was separately probed with antibody against p38 MAP kinase (bottom panel). As a positive control for Akt phosphorylation, the human leukemia cell line U937 was analyzed. CD34⁺ cells and CD34-depleted MNCs were purified as described and lysed in parallel. (B) Fresh patient samples were purified by Ficoll gradient and MNCs obtained prior to freezing (lanes 1 and 5) or frozen and then thawed. Samples after thawing were collected immediately (lanes 2 and 6) or incubated in serum-free medium with no cytokines for the indicated times (lanes 3-4, 7-8). K562 cells were used as positive control (lane 9).