(A) Binding profiles of the eIF1A aptamer using surface plasmon resonance. The aptamer RNA (apt) or the ‘N40’ random sequence RNA was immobilized on a sensor chip for Biacore2000 analysis. eIF1A solution was prepared and was injected at time 0 for 120 s. From the fitting of these sensorgrams by the instrument software BIAevaluation, the dissociation constant (Kd) of the aptamer-eIF1A interaction was estimated as 5.2 nM. (B) Pulldown assay of translation factors bound to eIF1A. The following samples were loaded on each lane: lane 1, 1 μl of HeLa cell extract; lane 2, coprecipitated sample by Ni-NTA agarose; lane 3, coprecipitated sample by Ni-NTA agarose with His-CAT; and lane 4, coprecipitated sample by Ni-NTA agarose with 6XHis-eIF1A. Separated and transferred proteins are detected by Western blotting using antibodies against eIF3 p66, eIF3 p110, rpS6, eIF2α and eIF2β (from upper to lower). These proteins bound with eIF1A specifically. (C) Pull-down assay from HeLa cell extracts using Ni-NTA agarose and 6XHis-eIF1A, with 0.3, 1, 3 or 10 μM N40 random RNA (N40) or 0.3, 1, 3 or 10 μM aptamer (labeled). Two components of eIF3, p66 and p110, plus rpS6, eIF2α and eIF2β bound to eIF1A. When aptamer was added to this pull-down reaction, the precipitation of eIF3 components and rpS6 was reduced, suggesting that the aptamer blocked the interaction of eIF1A with eIF3 and rpS6.