Effects of lysosome and proteasome inhibitors on TpoR cell-surface and total levels in JAK2 V617F cells. (A) Flow cytometric assessment of cell-surface HA-TpoR levels of Ba/F3 TpoR JAK2 V617F treated or not with the indicated inhibitors. Shown are averages of at least 3 independent experiments ± SD. Each experiment reflected analysis of 10 000 cells. **Two-tailed P < .01. (B) The indicated Ba/F3 cells were treated with 20 μg/mL cycloheximide for 20 hours or not and simultaneously treated with a combination of lysosome inhibitors (10μM, E64, 10μM pepstatin A, and 200μM leupeptin) or 10μM MG132 proteasome inhibitor. Shown are Western blot analyses for HA-TpoR and β-actin. (C) Western blot analysis with anti-HA antibodies and anti–β-actin antibodies of Ba/F3 TpoR JAK2 V617F cells treated with the indicated inhibitors for 20 hours. The densitometric ratios between the mature and immature TpoR bands were normalized to that of untreated cells.