Figure 6.

FBP1 outcompeted the binding of FBP2 to EV71 IRES. (A) The associated region in EV71 5′-UTR with FBP1 and FBP2. The linker region (636–745 nt) of the EV71 5′-UTR RNA probe was transcribed in vitro and biotinylated. RD cell lysates were incubated with a non-biotinylabeled RNA probe (lane 1) or a biotinylated RNA probe (lane 2). After being pulled down by Streptavidin beads, the protein complex was resolved in the SDS–PAGE (12%). Western blot was then performed to detect FBP1 and FBP2 in the pull-down complex. (B) The competition of FBP1 and FBP2 for IRES binding. The pull-down assay was performed here. The eluted proteins were subjected to SDS–PAGE (12%). Various amounts (µg) of FBP1 recombinant protein were added to compete with FBP2 of the cell lysate in interacting with the biotin-labeled EV71 5′-UTR RNA probe (EV71 1–745 nt) (lanes 2–6) and the linker region RNA probe (EV71 636–745 nt) (lanes 8–12). In the negative control, non-biotinylated RNA probes were applied the reaction (lanes 1 and 7). Antibodies against FBP1 and FBP2 were utilized in a western blot analysis.