Fig. 8: TpoR traffic in haematopoietic cell lines is dependent upon the presence of JAK2 V617F.

A HEL cells were transfected with TpoR alone or TpoR treated with Bafilomycin A1. 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 (middle and right panels) to identify areas with (yellow) and without FRET (red). (center) Overlay of FRET signal with donor TOLLES signal; (right) Inset shows an enlarged view with arrowheads indicating puncta with quenched FRET signal. The scale bar represents 10 μm. B The subcellular localization of TpoR was analyzed by live cell imaging and confocal microscopy. HEL cells were transfected with TpoR tagged with GFP and JAK2WT. Rab1A, Rab1B or Rab6 tagged with mCherry. The scale bar represents 10 μm. Arrowhead shows colocalization of TpoR W515L with respective Rab. C The subcellular localization of TpoR was analyzed by confocal microscopy. HEL cells were transfected with TpoR and scrambled siRNA or siRNA against Rab1A. Rab1B was overexpressed to ensure Golgi integrity. Samples were stained for organelle marker (green; LAMP1 or LC3) and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowhead shows colocalization of TpoR with LAMP1 and LC3. D The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in C 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 = 250–500 for LAMP1and 100–250 for LC3.