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. 2004 Sep 24;383(Pt 1):27–35. doi: 10.1042/BJ20040041

Figure 5. Nucleocytoplasmic shuttling of EEN/EA2.

Figure 5

(A) COS7 cells were transfected with wild-type untagged EEN/EA2 and immunostained with Ab2 antibody. Exogenous wild-type EEN/EA2 accumulated predominantly in the cytoplasm, suggesting that cytoplasmic localization was not attributed to epitope tagging. (B) Nucleocytoplasmic shuttling of EEN/EA2 in HeLa cells transfected with N-terminal FLAG-tagged EEN/EA2, immunodetected with anti-FLAG antibody and then probed with FITC-conjugated secondary antibody. (C) Schematic representation of the structural domains of EEN/EA2 and the various deletion mutants. The Lys-Arg-rich motif (KR1; amino acids 159–177) harbours the predicted bipartite basic NLS (amino acids 159–174). The coiled-coil region (amino acids 180–260) is linked to the C-terminal SH3 domain (amino acids 303–368) through the variable domain (amino acids 261–302), which is not well conserved in the endophilin family members. (D) Subcellular localization of EGFP–EEN/EA2 and its deletion mutants in HeLa cells. EGFP–EEN/EA2 (amino acids 1–179) also demonstrated midbody localization (arrowhead). Nuclear localization of the N-terminal region (amino acids 1–156) of EEN/EA2 suggested that the predicted bipartite basic NLS (amino acids 159–174) was dispensable for nuclear import. (E) Subcellular localization of FLAG-tagged NLS mutant of EEN/EA2 (ΔNLS). This indicated the dispensable nature of the predicted NLS on the regulation of nuclear import.