American Society of Hematology

Figure 3.

$FOXO3a is phosphorylated on Thr32 in Ba/F3 cells stably or inducibly expressing NPM-ALK. (A) FOXO3a phosphorylation in TonBaF.1 cells inducibly expressing NPM-ALK 24 hours after the addition of 2 μg/mL doxycycline. Uninduced samples were harvested from cells starved of IL-3 for 17 hours. +Dox 24hr samples were harvested from cells starved of IL-3 for 17 hours and induced with 2 μg/mL doxycycline for 24 hours. Whole-cell lysates were fractionated by SDS-PAGE and immunoblotted with phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reblotted with FOXO3a-specific antibody to confirm equal expression. (B) FOXO3a is constitutively phosphorylated in Ba/F3 cells stably transformed with NPM-ALK. Whole-cell lysates of MSCV- and MSCV-NPM-ALK-transduced Ba/F3 cells were harvested after a 17-hour IL-3 starvation, fractionated by SDS-PAGE, and immunoblotted with phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reblotted with FOXO3a-specific antibody to confirm equal expression. (C) NPM-ALK-induced phosphorylation of FOXO3a (Thr32) is dependent on PI 3-kinase activity. Whole-cell lysates of MSCV- and MSCV-NPM-ALK-transduced Ba/F3 cells were harvested after a 17-hour IL-3 starvation and 1-hour exposure to 0 μM, 20 μM, or 100 μM of the PI 3-kinase-specific inhibitor LY294002. Equal amounts of protein were fractionated by SDS-PAGE and immunoblotted with phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reblotted with FOXO3a-specific antibody to confirm equal expression. (D) Constitutive phosphorylation of FOXO3a in human NPM-ALK-positive ALCL cell lines. Whole-cell lysates of Karpas-299, SUDHL-1, and SR-786 cells were fractionated by SDS-PAGE and immunoblotted with a phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reprobed with a FOXO3a-specific antibody.$

FOXO3a is phosphorylated on Thr32 in Ba/F3 cells stably or inducibly expressing NPM-ALK. (A) FOXO3a phosphorylation in TonBaF.1 cells inducibly expressing NPM-ALK 24 hours after the addition of 2 μg/mL doxycycline. Uninduced samples were harvested from cells starved of IL-3 for 17 hours. +Dox 24hr samples were harvested from cells starved of IL-3 for 17 hours and induced with 2 μg/mL doxycycline for 24 hours. Whole-cell lysates were fractionated by SDS-PAGE and immunoblotted with phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reblotted with FOXO3a-specific antibody to confirm equal expression. (B) FOXO3a is constitutively phosphorylated in Ba/F3 cells stably transformed with NPM-ALK. Whole-cell lysates of MSCV- and MSCV-NPM-ALK-transduced Ba/F3 cells were harvested after a 17-hour IL-3 starvation, fractionated by SDS-PAGE, and immunoblotted with phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reblotted with FOXO3a-specific antibody to confirm equal expression. (C) NPM-ALK-induced phosphorylation of FOXO3a (Thr32) is dependent on PI 3-kinase activity. Whole-cell lysates of MSCV- and MSCV-NPM-ALK-transduced Ba/F3 cells were harvested after a 17-hour IL-3 starvation and 1-hour exposure to 0 μM, 20 μM, or 100 μM of the PI 3-kinase-specific inhibitor LY294002. Equal amounts of protein were fractionated by SDS-PAGE and immunoblotted with phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reblotted with FOXO3a-specific antibody to confirm equal expression. (D) Constitutive phosphorylation of FOXO3a in human NPM-ALK-positive ALCL cell lines. Whole-cell lysates of Karpas-299, SUDHL-1, and SR-786 cells were fractionated by SDS-PAGE and immunoblotted with a phospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reprobed with a FOXO3a-specific antibody.

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