Fig. S2.

eIF4E3 is not an importin 8 cargo. (A) Comparison of the surface charge distribution on the dorsal surface (Left) and cap-binding pocket (Right) of eIF4E and eIF4E3 in their apo form (PDB codes 2GPQ and 4B6V, respectively) (28, 30). Key residues are displayed. (B) Sequence alignment of human eIF4E and mouse eIF4E3, which differ by only six residues within the folded portion. Secondary structure elements are also shown. (C) GST pull-down assay using GST-eIF4E or GST-eIF4E3 as bait protein and importin 8 as prey. Clearly, importin 8 does not bind to eIF4E3 over the GST background. (D) In vitro nuclear import assays with GST-eIF4E3 demonstrated no nuclear entry upon addition of importin 8 compared with eIF4E (Fig. 1B). Confocal micrographs were single sections through the plane of the cells with a magnification of 63×. (Bottom) Quantification is shown. ****P < 0.0001.