FIGURE 1.

Constitutively activated and autophosphorylated Flt3-ITD rapidly undergoes both proteasomal and lysosomal degradation. A, Ton.32D/Flt3-ITD (Flt3-ITD) or Ton.32D/Flt3-WT (Flt3-WT) cells were cultured with 1 μg/ml DOX for 24 h to induce the Flt3-ITD or -WT expression, respectively. Ton.32D/Flt3-WT cells were then pretreated for 15 min with 20 ng/ml FL or left untreated as indicated. Cells were then treated with 50 μg/ml CHX and lysed after the indicated times. Cell lysates were subjected to immunoblot analysis with anti-Flt3 followed by reprobing with anti-β-actin as indicated. Relative expression levels of the mature (upper rows) and immature (lower rows) forms of Flt3, determined by densitometric analysis, are shown. B, after culture with DOX, Ton.32D/Flt3-ITD cells were pretreated for 30 min with 200 nm sorafenib or left untreated as the control as indicated and subjected to immunoblot analysis using antibodies against indicated proteins. C and D, after culture with DOX, Ton.32D/Flt3-ITD (Flt3-ITD) or Ton.32D/Flt3-WT (Flt3-WT) cells were pretreated for 2 h with 100 μm chloroquine (CQ) where indicated and with 50 ng/ml FL for Ton.32D/Flt3-WT. Cells were subsequently cultured for 30 min with or without 5 μm MG132 (MG) where indicated in the presence of 100 μm t-butoxycarbonyl-D-fmk. Cells were then treated with 10 μg/ml CHX for the indicated times and analyzed. E, MV4-11 cells were treated for 1 h with 25 μm chloroquine or 5 μm MG132 or left untreated as control (Cont.) as indicated in the presence of 50 μm benzyloxycarbonyl-VAD-fmk. Cells were then treated with 2 μg/ml CHX and analyzed.