Fig. 6: Unconventional secretion of TpoR requires RAB1A.

A The subcellular localization of TpoR was analyzed by live cell imaging and confocal microscopy. U2OS cells were transfected with TpoR tagged with GFP and JAK2WT or JAK2V617F or CALR ins5 or CALR del52. Rab1A, Rab1B or Rab6 tagged with mCherry. The scale bar represents 10 μm. Arrowhead shows colocalization of TpoR with respective Rab. B The subcellular localization of TpoR was analyzed by confocal microscopy. HEK cells were transfected with TpoR along with JAK2 V617F 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. C The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in (B) 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 < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 686–841 for LAMP1and 791–1208 for LC3. D HEK293 cells transfected with TpoR along with JAK2V617F and scrambled siRNA or siRNA against Rab1A were harvested 24 h after transfection and incubated with 50 ul of 10 mg/ml NHS-S-S-Biotin for 30 min. Cells were then lysed and incubated with streptavidin beads overnight at 4 degrees and then treated with Endo H and subjected to immunoblotting. Representative image of the blot is shown (n = 3).