JAK2 V617F expression leads to down-modulation of cell-surface and total TpoR protein. (A) Flow cytometric assessment of cell-surface HA-TpoR levels. Ba/F3 cells retrovirally transduced with the JAK2wt or JAK2 V617F were sorted to equivalent CD4 levels, as a truncated CD4 molecule is also coded by the bicistronic pMX retrovirus. Cells were subsequently transduced with bicistronic pMX viruses coding for TpoR or EpoR and GFP and were sorted for equivalent GFP levels. Shown are Ba/F3 cells maintained in medium supplemented with IL-3, Epo (for EpoR cells), and Tpo (for TpoR cells). Detection of cell-surface HA was performed using monoclonal anti-HA antibodies and R-PE–conjugated donkey F(ab′)2 anti–mouse IgG secondary antibody. At least of 10 000 events were analyzed for each flow cytometric measurement. (B) Confocal immunofluorescence analysis of cells expressing the indicated HA-TpoR and JAK2 constructs. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% saponin. After quenching of autofluorescence and blocking nonspecific labeling, detection of HA-TpoR was performed with monoclonal anti-HA antibodies and goat anti–mouse IgG linked to Alexa-568 secondary antibody. Bottom right panel: Image with artificially increased luminosity (+40%) and contrast (+40%). (C) Quantitative PCR determining the levels of expression of TpoR mRNA in the indicated cell lines. (D) Western blot analysis with anti-HA antibodies, anti-JAK2, and anti–β-actin antibodies of Ba/F3 cells expressing the indicated TpoR and JAK2 constructs and maintained in medium supplemented with IL-3 (cells shown in panel A bottom right panel, +IL-3). Normalized ratio mature/immature TpoR bands were calculated after dividing the densitometric measurement of the intensities of mature and immature bands (ratio) and normalizing to the ratio in Ba/F3 wtTpoR cells.
