Signal transduction patterns of mutated and wt-Flt3 in primary AML cells and cell cycle analysis. (A) PB was taken before start with PKC412. MNCs were separated and treated with FL (100 ng/mL), SCF (100 ng/mL), 100 nM PKC 412, imatinib mesylate (1 μM) and in combination for 15 minutes as indicated. Whole-cell lysates were analyzed by Western blot. Constitutive tyrosine phosphorylation of FLT3 and STAT-5 in primary AML blasts harboring mutated FLT3 using specific antiphosphotyrosine antibodies for FLT3 and STAT-5 was detected. Akt and Erk1/2 activation were investigated using specific antiphosphoserine-Akt and antiphosphotyrosine-Erk antibodies, respectively. Further, FL or SCF effects on the investigated signaling pathways are demonstrated. To control equal loading, the blot was stripped and reprobed. (B) Primary MNCs obtained from AML patient PN 1 were maintained in RPMI 1640 without any growth factor supplementation and incubated with or without PKC412 (50 nM), FL (100 ng/mL), or in combination for 90 hours as indicated, and cell cycle analysis was performed.