EPHB4 promotes AML survival through AKT signaling. (A) K562 cells were transduced with siRNA against EPHB4 and analyzed for EPHB4, pAKT, and total AKT at 48 hours. pAKT levels were reduced in EPHB4 knockdown cells as measured by immunoblotting, whereas total AKT levels were unchanged. (B) K562 cells were co-transduced with siRNA against EPHB4 and/or the constitutively active form of AKT (myr-AKT). Expression of myr-AKT restores cell growth and survival after EPHB4 knockdown. The average counts from 3 separate experiments are displayed, and error bars represent the standard error of the mean. (C) Levels of AKT activation in cells treated in Figure 3B as measured by phosphor-flow for pAKT. (D) K562 cells were treated with MAb131, and pAKT was measured by flow cytometry at various time points. At 30 minutes after treatment, a transient increase in pAKT was observed due to EPHB4 activation by MAb131, followed by a decrease in pAKT as EPHB4 activity was lost. (E) Primary AML blasts (unstimulated) treated with MAb131 show decreased pAKT after treatment with MAb131.