CALR mutants modify TpoR stability, maturation, and trafficking. (A) The expression of CALR in calr^−/− MEFs transduced with retroviral constructs expressing TpoR and CALR wild-type or mutants was assessed by immunofluorescence (top panel) and by western blotting after cycloheximide (30 μg/mL) treatment (bottom panel). (B) Maturation state of TpoR in Ba/F3-TpoR cells in the presence of CALR wild-type or mutants was assessed by western blotting upon endoglycosidase H treatment or not. (B) Subcellular fractionation of CALR wild-type and mutant del52 proteins in calr^−/− MEF TpoR cells. The distribution patterns of CALR and GRP78 were shown in the diagram and the blots. (C) Confocal immunofluorescence microscopy was performed in previously described calr^−/− MEF cell lines and stained with anti-CALN (Calnexin), anti-CALR, and 4,6 diamidino-2-phenylindole. (D) Maturation state of TpoR in Ba/F3-TpoR cells in the presence of wild-type or mutant CALR was assessed by western blotting upon endoglycosidase H treatment or not. (E) Western blot analysis of immunoprecipitated N-terminal HA-tagged TpoR present on cell surface, in Ba/F3-TpoR cells expressing wild-type or mutant CALR. All data are normalized, C being the amount per volume of subfraction, and Ci the corresponding value in the pooled subfractions.