Immuno-electron microscopy of CALR. (A) In BaF3 cells, Flag-tagged CALR-del52 localized in both the cis-Golgi (cG) and trans-Golgi (tG) compartments (blue arrows), as well as vesicles distributed between the trans-Golgi network and the plasma membrane (blue circles), suggesting it follows the secretory pathway to the cell surface. (B) By contrast, Flag-tagged CALR WT predominantly localized in the ER and nuclear heterochromatin (yellow arrows) and was less frequent in the Golgi network (cG, tG). (C) In cytokine-independent, proliferating clustered regularly interspaced short palindromic repeats (CRISPR)-modified BaF3 TpoR Calr^mut cells, which expressed both CALR-del52 as well as endogenous CALR, anti–N-terminus labeling was frequently observed at the plasma membrane (blue circles), suggesting CALR secretion is maintained in this cell line. (D) In control CRISPR BaF3 TpoR Calr^wt cells, endogenous CALR localized mostly in the nucleus (N), perinuclear space, ER (yellow arrows), and the Golgi (G) network. (E) In primary Calr ^del52/WT KI mouse bone marrow cells, N-terminus–labeled CALR could be detected within the Golgi network (not shown), as well as at the plasma membrane (blue circles), suggesting secretion of CALR is maintained in these cells. (F) In control Calr^WT/WT mouse bone marrow cells, endogenous CALR was mostly localized at the ER and the nucleus (N). When using a mutant-specific anti-CALR antibody labeling could be detected in the Golgi network of CRISPR-modified BaF3 TpoR Calr^mut cells (G) (blue arrows) but also at the plasma membrane (H), including associated with ectosomes (blue circles). (I) Similarly, the mutant-specific antibody identified CALR-del52 in the secretory pathway of Calr^del52/WT KI mouse bone marrow cells (blue arrows and circles). Gold particle size is on average 0.8 nm in panel A and 6 or 10 nm in panels B to I. Scale bars represent 500 nm (A,F) and 200 nm (B-E,G-I). m, mitochondria.