Endos is a cytoplasmic protein, and tags can perturb its nucleocytoplasmic distribution. (A) Fluorescence images of fixed D-Mel cells expressing the indicated tagged forms of Endos. Note that GFP, RFP, and Myc (six copies), which are larger tags, make Endos more nuclear than cytoplasmic. Conversely, Flag (three copies) and the PrA, which are smaller tags, keep Endos in the cytoplasm. (B) Subcellular fractionation and WB show the relative amounts of cytoplasmic and nuclear proteins. MEK and histone H3 are controls as cytoplasmic and nuclear proteins, respectively. (C) Inhibition of Crm1-dependent nuclear export does not cause a strong nuclear accumulation of Endos. Left: D-Mel cells were treated with 50 nM LMB or 0.1% ethanol (control) for 4 h before fixation, immunostaining and DNA staining with DAPI. Scale bar: 5 µm. Right: Quantifications of the nuclear/cytoplasmic ratio of signal intensities (mean ± SD). (D) Endos is mainly cytoplasmic throughout the cell cycle. IF showing the localization of endogenous Endos in the different phases of the mitotic cell cycle. Scale bar: 5 µm. C, cytoplasmic; N, nuclear.