Fig. 2.

Binding of eIF3 subassemblies to the HCV IRES IIIabc domain. (A) Native agarose gel showing binding of the PCI/MPN octamer to the HCV IRES IIIabc domain, schematically drawn to the right. The nanomolar concentrations of the octamer are listed. Lane 1 shows IIIabc RNA binding to natively purified human eIF3 as a control (lane 1). Fluorescent IIIabc RNA was used at 20 nM in concentration, and the reactions were carried out in the presence of 2 μM tRNA to prevent nonspecific binding. (B) Native agarose gel showing binding of the PCI/MPN octamer to the 40S ribosomal subunit, monitored by UV absorbance of the 40S subunit rRNA. The nanomolar concentrations of the PCI/MPN octamer are given, and the 40S ribosomal subunit was used at a concentration of 10 nM. Shown as a control, 40S subunit binding to natively purified human eIF3 (lane 1). (C) Coomassie blue-stained SDS gel showing PCI/MPN octamer affinity-purified using MBP-tagged eIF1A, or using MBP-tagged eIF1. MW markers are shown in kilodaltons. Arrows indicate the position of MBP-eIF1A (lanes 2, 4) and MBP-eIF1 (lanes 5, 7). Binding and wash conditions prevent nonspecific binding of the PCI/MPN octamer to the beads (lane 8). (D) Coomassie blue-stained SDS gel showing reconstituted eIF3 10-mer affinity-purified using the FLAG-tagged central domain of eIF4G. MW markers are shown to the left. Arrow indicates the position of subunit eIF3b. GroE copurified with eIF3d (asterisks), as determined by MS analysis. Antibody heavy (H) and light (L) chains are marked. The concentration of KCl used in the washes (200 mM) prevents nonspecific binding to the anti-FLAG beads (Fig. S4).