4E-BP1 and p70S6 kinase are phosphorylated in primary AML cells. (A) Cell lysates (106 cells) from 7 AML patients and normal unstimulated and stimulated (GM-CSF, SCF, and IL-3 for 24 hours) CD34⁺ HPCs were analyzed by Western blotting with specific antibodies to evaluate the phosphorylation status of 4E-BP1 and p70S6K. The phosphorylation of 4E-BP1 can also be visualized by a shift in the apparent molecular weight of the protein. Results shown are representative of 23 fresh AML samples tested. (B) Fresh AML cells from 4 patients were incubated with RAPA (10 nM) for 24 hours and analyzed by Western blotting with the indicated antibodies.