<p>(A) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours prior to lysis. Blots were probed against STAT5 pTyr694, AKT pSer473 and MAPK pThr202/pTyr204 stripped and subsequently reprobed against total STAT5, AKT and MAPK. One out of three independent representative experiments is shown. (B) Semi-quantitive analysis of (p)STAT5 band intensity was performed to calculate the ratio between pSTAT5 and total STAT5. Values are expressed as mean +/− S.D. of three independent experiments. (*) indicates significance to FLT3-WT expressing cells. The signal intensity of Ba/F3 MIY expressing cells has been subtracted from FLT3-WT and FLT3 mutant values. (C) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours prior to lysis. Equal amount of whole cell lysates were used in a chemiluminescent ELISA assay detecting phosphorylation of FLT3 receptor at Tyr591. Values are expressed as mean +/− S.D. of three independent experiments. Grouped analysis of FLT3 mutants revealed significant differences between FLT3-WT expressing cells and FLT3-WT-like cell lines (p = 0.045) as well as FLT3-WT expressing cells and FLT3-ITD cell lines (p = 0.005). Further the group of FLT3-ITD cell lines showed significant differences compared to FLT3-WT-like mutants (p = 0.008) and FLT3-TKD cell lines (p≤0.001). (*) indicates significance of grouped analyses and individually compared to FLT3-WT expressing cells. The signal intensity of Ba/F3 MIY expressing cells has been subtracted from FLT3-WT and FLT3 mutant values. RLU: Relative light units. (D) 4×10<sup>4</sup> cells/ml stably transduced with indicated constructs, were cultured in presence or absence of 2 µM MK 2206 and/or 10 ng/ml IL-3. Cells were counted after 72 hours by trypan blue exclusion. (*) indicates significant growth reduction by MK 2206 compared to untreated cells. Proliferation with MK 2206 is shown in relation to untreated cells. Control indicates for Ba/F3 MIY expressing cells treated with 2 µM MK 2206 in the presence of IL-3. Values are expressed as mean +/− S.D. of three independent experiments.</p
