Fig. 5: CALR mutations block unconventional traffic of TpoR.

A The subcellular localization of TpoR was analyzed by confocal microscopy. HEK293 cells were transfected with TpoR along with JAK2 WT and CALR ins5 or del52 and stained for organelle marker (green; LC3) or lysotracker and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowheads indicate colocalization of TpoR with lysotracker. B The signal intensities of TpoR puncta and lysotracker or LC3 puncta in A were calculated as gray values and the percentage colocalization of TpoR was calculated in each case. Data represents percentage of colocalization ±SEM. Error bars represent SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 19–209 for lysotracker and n = 38–143 for LC3. C HEK293 cells expressing TpoR along with JAK2 WT and CALR ins5 or del52 were lysed 24 h post-transfection and treated with Endo H or PNGase F and subjected to Western blot analysis. Representative image of the blot is shown (n = 3). D U2OS cells were transfected with SRAI-TpoR, JAK2 WT, CALR del52. Imaging of TOLLES-YPet signal was performed under a confocal microscope. (Left) Ratiometric FRET (Intensity of TOLLES/ Intensity of YPet) approach was used to generate the rainbow RGB images. The ratiometric FRET image (left panel) has been represented; overlay of the donor TOLLES signal (red) and FRET signal (green) has been represented (right panel) to identify areas with (yellow) and without FRET (red). (right) Overlay of FRET signal with donor TOLLES signal. The scale bar represents 10 μm. E HEK293 cells expressing TpoR along with JAK2 WT and CALR del52 mutants H170A or Y109F/D135L were lysed 24 h after transfected and co-immunoprecipitated using anti-HA antibody followed by immunoblotting with an anti-Flag antibody for the CALR del52 double mutants. F The subcellular localization of TpoR was analyzed by confocal microscopy. HEK293 cells were transfected with TpoR along with JAK2 WT and CALR del52 mutants H170A or Y109F/D135L and stained for organelle marker lysotracker (green) and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowhead represents colocalization of TpoR with lysotracker. G The signal intensities of TpoR puncta and lysotracker in (F) were calculated as gray values and the percentage colocalization of TpoR was calculated in each case. Data represents percentage of colocalization ± SEM. Error bars represent SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 209–1295 for lysotracker.