Fig. 5. Mutation of a putative NLS in N-FAG changes its localisation from nuclear to cytoplasmic. (A) Schematic representation of eIF4GI showing the basic region that may act as a nuclear localisation signal and the individual sequences used in this study. (B) HeLa cells were transfected with plasmids encoding the wild-type or ₅₁₃KRRRK_517-513AAAAA₅₁₇ mutated myc-tagged eIF4GI sequences indicated. 16 hours after transfection, total cell lysates were prepared and proteins resolved by SDS-PAGE. eIF4GI and its N-terminal cleavage fragments were visualised by immunoblotting using anti-myc antiserum; molecular mass markers are shown on the left. Expression of eIF4GIf_AAAAA is only detectable at extremely long exposures (data not shown). (C) HeLa cells were probed with anti-myc antibody followed by goat anti-mouse IgG conjugated to FITC (green) to visualise the localisation of wild-type N-terminal apoptotic cleavage fragments of eIF4GI, or those in which a basic sequence was mutated to alanines, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.