eIF4GI mediates the Ago2-cap association.

<p>(<i>A</i>) Identification of a translation initiation factor capable of augmenting the Ago2-cap association. WCEs from 293FT cells co-transfected with plasmids expressing myc-Ago2 and Flag-tagged translation factors (eIF3c, eIF4AI, eIF4E, eIF4GI and PABP) were subjected to cap-pulldown assays. The expression levels of the transfected genes (lanes 1–6), their cap-associations (lanes 7–12), and various proteins from WCEs or from the resin-bound fractions were monitored by Western blotting with the indicated antibodies. (<i>B</i>) The eIF4GI-dependent cap-association of Ago2. WCE from HeLa cells transfected with si-Control or si-eIF4GI were applied to cap-pulldown assays. The knock-down efficiency of siRNAs and the resin-bound proteins were monitored using antibodies described. (<i>C</i>) Determination of a domain in eIF4GI responsible for augmenting the Ago2-cap association. WCEs from 293FT cells transiently expressing myc-Ago2 and Flag-tagged fragments encoding the N-terminal, middle or C-terminal regions of eIF4GI (depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#pone-0055725-g005" target="_blank">Figure 5A</a>) were subjected to cap-pulldown assays. The resin-bound Ago2 (upper panel), the ectopically expressed Ago2 and eIF4GI fragments (lower panel), GAPDH and eIF4E proteins were detected using the indicated antibodies.