FLT3-ITD protein expression is downregulated by bortezomib and precedes cell death. (A-B) FLT3-ITD protein level is downregulated by bortezomib (Bort). MV4-11 or MOLM14 or were treated 24 hours with bortezomib at different concentrations. Total cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the appropriate antibodies. (C) MOLM-14 cells were treated 24 hours with 10 nM bortezomib and then analyzed by western blot. (D) FLT3-ITD–positive patient samples were exposed 24 hours with bortezomib at different concentrations before immunoblotting analysis. (E-F) FLT3-ITD downregulation precedes cell death. MV4-11 cells were treated with 10 nM bortezomib for 8 to 24 hours before cell lysis and western blot analysis using the appropriate antibodies. FLT3 protein expression levels were determined by densitometry analysis (Image J software). In parallel, cell death was evaluated by Annexin-V staining and flow cytometry analysis. Cell death (% of maximal effect) represents the ratio ([Annexin-V⁺ cells t = x*hours/Annexin-V⁺ cells t = 24 hours] ×100).