ESE-1 contains a basic AA-rich NLS sequence. (A) Diagram of ESE-1 protein and sequence of the ESE-1 region containing putative ESE-1 NLS motifs 1-5 (AAs 236-268). Colored boxes indicate ESE-1 domains. A red line below the diagram indicates localization of AAs 236-268, with the sequence expanded below. AA sequences for putative ESE-1 NLS motifs 1-5 are aligned with the ESE-1 236-268 AA sequence. Red letters - AAs potentially required for nuclear localization. Red line above diagram - localization of putative NLS6 motif. (B) Map and AA sequence of GFP-NLS1-SAR. GFP (green box, not to scale) followed in-frame by NLS1 (red letters). The AA sequence for all but the first two amino acids in the ESE-1 SAR domain (blue box) is fused to the carboxy-terminal end of NLS1, followed by ESE-1 flanking sequence (white box). All remaining NLS fusion proteins were constructed the same way. (C) Representative fluorescence microscopy images of MCF-12A cells transiently transfected with GFP-NLS1-SAR (panel 1), GFP-NLS2-SAR (panel 2), GFP-NLS3-SAR (panel 3), GFP-NLS4-SAR (panel 4), GFP-NLS5-SAR (panel 5), or GFP-SAR (panel 6) (D) Left panel - AA sequence alignments among putative ESE-1 NLS6 and ETS-1, ELK-1, ER71 NLS motifs. Superscripted numbering indicates the position of each AA sequence within its parental protein. Right panel - representative fluorescence microscopy images of MCF-12A cells, transiently transfected with the GFP-NLS6-SAR. (E) Left panel - diagram of GFP-ESE-1ΔDBD. In-frame deletion of ESE-1 DBD is represented by a V-shaped line. Right panel - representative MCF-12A cells transiently transfected with the GFP-ESE-1ΔDBD construct imaged by fluorescence microscopy. In all images white dashed lines represent cell outlines.