ESE-1 contains two separate CRM-1 dependent NES motifs. (A) Block diagram of ESE-1 protein with domains indicated by labeled colored boxes. NES1 and NES2 are represented by black lines above the diagram. (B) Schematic of GFP-NES1-SAR and GFP-NES2-SAR proteins, constructed as described in Figure 1B. Green box - GFP. White box - NES1 or NES2. Critical NES leucine and isoleucine residues are shown in red. Blue box followed by white box - ESE-1 fragment 190-239 containing the SAR domain (AAs 190-229). (C) Fluorescence imaging of transiently expressed GFP-NES1-SAR (± leptomycin B; panels 1, 2, 3, & 4), GFP-NES1Mut-SAR (panels 5 and 6), GFP-NES-2-SAR (± leptomycin B; panels 7, 8, 9, & 10), and GFP-NES2Mut-SAR (panels 11 & 12) in living MCF-12A cells. All transfectants were DAPI-stained to identify the nuclei and some GFP-NES1-SAR and GFP-NES2-SAR transfectants were treated with leptomycin B prior to microscopy. Upper panels - GFP fluorescence (green). Lower panels - overlays of GFP (green) and DAPI (blue) fluorescence. Although only one or two representative cells are shown for each transfectant population, a ~30% transfection efficiency was consistently achieved per 106 cells transfected and ~105 green fluorescent cells were present following each transfection. White dashed lines represent cell outlines.