Enhanced nuclear localization of CHC lacking a C-terminal region containing a trimerization domain. (a) Part of CHC833-1406 is localized in nuclei. HA-tagged full-length CHC or CHC833-1406 was expressed in H1299 cells fixed with 4% paraformaldehyde. The fixed cells were sequentially incubated with anti-HA antibody and AlexaFluor-594-conjugated secondary antibody, and subjected to immunofluorescent microscopic analysis. Nuclei were counterstained with propidium iodide (PI). (b) Subcellular localization of CHC in cells was quantified by scoring fluorescent signals from confocal immunofluorescent microscopic analysis. The percentage of cell population of cellular localization was calculated using more than 100 cells in three independent experiments. The percentage of cells, predominantly localized in cytoplasm (open bars), equally localized in cytoplasm and the nucleus (hatched bars), and predominantly localized in the nucleus (black bars) is shown, respectively. (c) Localization of CHC is not affected by treatment with leptomycin B (LMB). H1299 cells were transfected with 400 ng of HA-tagged CHC or 400 ng of GFP-tagged p53 fused to nuclear export signal (NES) construct. The next day, cells were treated with 10 nM LMB for 24 h and analysed by confocal immunofluorescent microscopy. Nuclei were counterstained with PI. (d) Subcellular localization of CHC in cells treated with LMB was quantified by scoring fluorescent signals from immunofluorescent microscopic analysis as described in (b).