V617F mutation favors nuclear translocation of JAK2 in K562 and early CD34⁺ progenitors isolated from BM of MPN patients. This translocation is not observed in differentiated cells. (A) CIF microscopy images of K562 cells stably transfected with pMSCV-JAK2 (top-row images) or pMSCV-JAK2V617F (second- and third-row images) Western blotting of cytoplasmic (C) and nuclear (N) fractions (bottom) confirm that JAK2V617F is more abundant than JAK2 in K562 nuclei. (B) CIF images of BM cells from an MPN patient with wt JAK2 (top row) and an MPN patient with JAK2V617F (bottom row) confirm a nuclear increase of the mutated protein. (C) CIF demonstrates a predominantly nuclear accumulation of JAK2V617F in CD34⁺ cells isolated from BM of 1 essential thrombocythemia (ET), 1 polycythemia vera (PV), and 1 early primary myelofibrosis (PMF) (left panels). Western blotting of cytoplasmic (C) and nuclear (N) extracts (right) confirm the data. (D) CIF images of CD15⁺, CD41⁺, CD71⁺ cells isolated from a JAK2V617F-positive MPN patient (PV, JAK2 allele burden 71%) and Western blotting of cytoplasmic (C) and nuclear (N) extracts (right). DAPI, 4,6-diamidino-2-phenylindole; Anti-JAK2 (Cell Signaling Technology) monoclonal antibody; anti-tubulin and anti-laminin A (Sigma-Aldrich) monoclonal antibodies.