Figure 6  eEF1A binds to the CSFV internal ribosome entry site (IRES). (A) eEF1A as well as NS5A interacts with the CSFV IRES. HEK293T cells were transfected with pMyc-eEF1A or pMyc-NS5A. The cell lysate was incubated with the biotinylated CSFV IRES or porcine reproductive and respiratory syndrome virus (PRRSV) 3ʹ-UTR, followed by incubation with streptavidin beads. The bound proteins were analyzed by immunoblotting using the anti-Myc monoclonal antibody (MAb) (1:1000); (B) eEF1A interacts with the CSFV IRES in a dose-dependent manner. HEK293T cells grown in the 6-well plate were transfected with an increased amount of pMyc-eEF1A (0, 1, 2 and 4 μg), followed by streptavidin pulldown assay as described above; (C) Competitive RNA pulldown assay. HEK293T cells grown in the 6-well plate were transfected with the indicated pMyc-eEF1A (4 μg). The cell lysate was incubated with an increased amount of the IRES RNA (0, 0.75, 1.5 and 3.0 μg) for 1 h at 4 °C and incubated with the biotinylated IRES RNA and streptavidin beads. The bound proteins were subjected to immunoblotting using the anti-Myc MAb.