Western immunoblot analyses of mutant-TK Ba/F3 cells as a cell model for activated AKT-signaling. (A) Transfection of leukemia-driving mutant-KIT, FLT3 or BCR-ABL1 isoforms into a hematopoietic IL3-dependent Ba/F3 cell line leads to global increase of AKT phosphorylation as assayed by immunoblotting of whole cell extracts. Phosphorylation of Ser473 and Thr308 increases in response to IL3 stimulation - and further augments in cells transfected with a gain-of-function mutant-TK. Tubulin blotting is used as loading control. (B) An immunoblot experiment with Ba/F3 cells transfected with a FLT3 D836V mutation treated with NVP-BGT226 or NVP-BEZ235 and probed for phosphorylation patterns of AKT is shown. Jurkat cells are used as positive controls. Jurkat cells treated with PI3K inhibitors (Wortmannin, LY294002) serve as negative controls for AKT phosphorylation. Actin blotting serves as a loading control.