JAK2 targets the membrane of Baf3 and HEL cells and is excluded from the nucleus. (A) Confocal fluorescent images in Baf3/EPOR cells after transient transfection with JAK2WT-mCitrine. The nucleus is labeled with Hoechst. (B) Confocal images of Baf3/EpoR cells transiently transfected with JAK2V617F-mCitrine (green fluorescence). Cells were also stained with anti-JAK2 (Cy5, red fluorescence) and Hoechst. (C-D) Analysis of endogenous JAK2 distribution in HEL and K562 cells labeled with Hoechst and anti-JAK2 antibodies. Red lines indicate lines drawn across individual cells in the confocal image, with matching plots below reporting fluorescence intensity. (E) Analysis of nuclear and cytosol extracts from HEL cells, subjected to SDS-PAGE, Western blotting and immunoblotting with JAK2, tubulin, and phospho-histone 3 antibodies.(F-G) Confocal fluorescent images in HEL and K562 cells after transient (HEL) or stable (K562) transfection with JAK2V617F-mCitrine (HEL) or JAK2WT-mCitrine (K562) (green fluorescence). Nuclei were stained with Hoechst and endogenous JAK2 was stained with an anti-JAK2 antibody detected by a secondary antibody coupled to a Cy5 dye. Confocal fluorescent images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× oil objective. ZEN 2009 5.5 SP1 software was used for both image acquisition and processing. Before imaging, treated cells were mounted on slides using ProLong Gold antifade reagent (Invitrogen).