V617F mutation causes up-regulation of LMO2. The JAK2 inhibitor AG490 replaces JAK2 into cytoplasm and restores LMO2 levels. (A) CIF images show the redistribution of JAK2 and the replacement in the cytoplasm in V617F expressing K562 after PMA differentiation. (B) CIF images (images) and Western blotting of cytoplasmic (C) and nuclear (N) extracts (right) show the redistribution of JAK2 and the replacement in the cytoplasm in the majority of V617F expressing K562 (bottom right) but not in wt cells (top right) after AG490 incubation. (C) Quantitative RT-PCR reveals that V617F mutation strongly up-regulates LMO2 expression in K562 and in CD34⁺ cells (left graphs). The addiction of AG490 progressively and completely restore LMO2 levels in V617F expressing K562 (right graphs) after 3 hours' incubation. Error bars represent standard deviation. DAPI, 4,6-diamidino-2-phenylindole; Anti-JAK2 (Cell Signaling Technology) monoclonal antibody; anti-tubulin and anti-laminin A (Sigma-Aldrich) monoclonal antibodies.