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Fig. 3. All isoforms of exogenous myc-tagged N-FAG are localised to the nucleus. (A) Schematic representation of eIF4GI outlining the individual N-terminal sequences used for expression in this study. (B) HeLa cells were transfected with plasmids encoding myc-tagged isoforms of the N-terminal domains of eIF4GI. Sixteen hours after transfection, total cell lysates were prepared and proteins resolved by SDS-PAGE. eIF4GI and its N-terminal cleavage fragments were identified by immunoblotting using anti-myc antiserum; molecular mass markers are shown on the left. Owing to differences in expression levels and to aid visualisation, extracts from cells transfected with N-FAGdc, N-FAGb, N-FAGa and Nta were all diluted 1:2 with sample buffer; despite this, expression of Ntf is only detectable at extremely long exposures (data not shown). (C) Anti-myc antibody followed by goat anti-mouse IgG conjugated to FITC (green) was used to visualise the localisation of the tagged isoforms of the N-terminal domain of eIF4GI expressed in HeLa cells, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.
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