Effects of JAK2wt and JAK2 V617F on TpoR protein processing and recycling. (A) Recycling of biotinylated cell-surface TpoR after ligand addition and removal was assessed after cell-surface biotinylation, as described in “Cell-surface biotinylation followed by TpoR cell-surface immunoprecipitation, recycling assays.” After cell-surface biotinylation, cells were treated or not with Tpo for the indicated times; then Tpo was removed for 15 minutes. Cells were surface immunoprecipitated by incubating cells at 4°C with anti-HA for 1 hour. After extensive washing to remove unbound anti-HA, cells were lysed in 1% NP40 buffer. Immune complexes of anti-HA and surface proteins were precipitated by Protein G-Sepharose beads. Samples were denatured by boiling in Laemmli buffer and analyzed by Western blotting with anti-HA or anti-biotin antibodies coupled to HRP to detect biotinylated (cell-surface) proteins. Vertical line(s) have been inserted to indicate a repositioned gel lane. (B) Confocal imaging of the TpoR in the indicated Ba/F3 cells permeabilized with saponin in the presence or absence of an overexpressed JAK2wt or JAK2 V617F. Transferrin/Alexa-488 (left panel) was absorbed, internalized, and recycled by the cells for 25 minutes at 37°C. After paraformaldehyde fixation, staining for the receptor was performed using anti-HA antibody and a secondary antibody labeled with Alexa-568. Ba/F3 cells expressing TpoR alone or with JAK2 show partial colocalization between TpoR and transferrin. In contrast, Ba/F3 TpoR JAK2 V617F shows no colocalization of TpoR with transferrin. TpoR expressed in JAK2 V617F cells exhibits an intracellular distribution and low cell-surface localization.