Toeprinting analysis of VLR insertion/deletion mutant IGR IRESs.

<p>Dicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in (A) RRL without drug treatment [−], or under treatment with edeine [E] or cycloheximide [C] or (B) in the absence [−] or presence [+] of purified salt-washed HeLa 80S ribosomes (100 nM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051477#s2" target="_blank">Materials and Methods</a> section. Primer extension analysis was performed using PrEJ69 in the presence of α- [³²P]-dATP. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The sequencing reactions for the wild-type IRES are shown on the <i>left</i>, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the <i>right</i>. Shown are representative gels from at least two independent experiments.