Fig. 2. Exogenously expressed myc-tagged isoforms of eIF4GI are localised to the cytoplasm. (A) Schematic representation of eIF4GI showing sites of alternative translation initiation and binding sites of other components of the translation initiation machinery (Bradley et al., 2002; Byrd et al., 2002; Morley, 2001). Sites of cleavage by caspase-3 and the picornaviral L and 2A proteases are indicated. Black lines indicate individual sequences used for expression in this figure. (B) Total cell lysates were prepared from HeLa cells transfected with plasmids containing eIF4GI cDNAs and resolved by SDS-PAGE. Expressed proteins were identified by immunoblotting using the 9E10 monoclonal antibody to the c-myc epitope; molecular mass markers are shown on the left. (C) Anti-myc monoclonal antibody (9E10) followed by goat anti-mouse IgG conjugated to FITC (green) was used to visualise the localisation of the tagged isoforms of eIF4G expressed in HeLa cells, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.