Figure 2. AKT is phosphorylated on... | American Society of Hematology

$Figure 2. AKT is phosphorylated on Ser473 in N/A-Ba/F3 cells in a PI 3-kinase-dependent manner. (A) Time-dependent AKT phosphorylation in TonBaF.1 cells inducibly expressing NPM-ALK 24 hours and 48 hours after the addition of 2 μg/mL doxycycline. Uninduced samples were harvested from cells starved of IL-3 for 17 hours. +Dox 24-hour and +Dox 48-hour samples were harvested from cells starved of IL-3 for 17 hours and induced with 2 μg/mL doxycycline for 24 hours and 48 hours, respectively. Whole-cell lysates were fractionated by SDS-PAGE and immunoblotted with phospho-AKT (Ser473)-specific antibody. The membrane was stripped and reblotted with AKT-specific antibody to confirm equal expression. (B) AKT is constitutively phosphorylated in Ba/F3 cells stably transformed with NPM-ALK. Whole-cell lysates of MSCV- and MSCV-NPM-ALK-transduced Ba/F3 cells were harvested after a 17-hour IL-3 starvation, fractionated by SDS-PAGE, and immunoblotted with phospho-AKT (Ser473)-specific antibody. The membrane was stripped and reblotted with AKT-specific antibody to confirm equal expression. (C) NPM-ALK-induced phosphorylation of AKT (Ser473) is PI 3-kinase-dependent. Whole-cell lysates of MSCV- and MSCV-NPM-ALK-transduced Ba/F3 cells were harvested after a 17-hour IL-3 starvation and 1-hour exposure to 0 μM, 20 μM, or 100 μM PI 3-kinase-specific inhibitor LY294002. Equal amounts of protein were fractionated by SDS-PAGE and immunoblotted with phospho-AKT (Ser473)-specific antibody. The membrane was stripped and reblotted with AKT-specific antibody to confirm equal expression.$

Figure 2.

AKT is phosphorylated on Ser473 in N/A-Ba/F3 cells in a PI 3-kinase-dependent manner. (A) Time-dependent AKT phosphorylation in TonBaF.1 cells inducibly expressing NPM-ALK 24 hours and 48 hours after the addition of 2 μg/mL doxycycline. Uninduced samples were harvested from cells starved of IL-3 for 17 hours. +Dox 24-hour and +Dox 48-hour samples were harvested from cells starved of IL-3 for 17 hours and induced with 2 μg/mL doxycycline for 24 hours and 48 hours, respectively. Whole-cell lysates were fractionated by SDS-PAGE and immunoblotted with phospho-AKT (Ser473)-specific antibody. The membrane was stripped and reblotted with AKT-specific antibody to confirm equal expression. (B) AKT 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-AKT (Ser473)-specific antibody. The membrane was stripped and reblotted with AKT-specific antibody to confirm equal expression. (C) NPM-ALK-induced phosphorylation of AKT (Ser473) is PI 3-kinase-dependent. 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 PI 3-kinase-specific inhibitor LY294002. Equal amounts of protein were fractionated by SDS-PAGE and immunoblotted with phospho-AKT (Ser473)-specific antibody. The membrane was stripped and reblotted with AKT-specific antibody to confirm equal expression.

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