The effect of eRF1 depletion on termination and recoding.

<p>A. eRF1 transcript levels measured using quantitative real-time PCR. Results show eRF1 transcripts within RNA isolated from non-transfected HEK293T cells (‘none’), and eRF1 transcripts within RNA isolated from those transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The mean ± standard deviation (SD) for six replicates is shown. B. Immunoblot of eRF1 in protein extracts from non-transfected HEK293T cells (‘none’), or cells transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The ratios were calculated after normalisation to β-actin in each case and compared with the non-transfected control (1.0). Raw data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s001" target="_blank">S1 Fig.</a> C. Readthrough at a UGA test context (UGACAG). A dual luciferase construct with the two reporters in the same frame and the test stop signal separating them was co-transfected with the control and α-eRF1 shRNAs. Readthrough at the test stop signal was determined in each case (α-eRF1 and − control.) The mean ± SEM for 12 replicates from three individual experiments is shown. D. Effect of depletion of eRF1 on +1 frameshift efficiency at the human antizyme frameshift element. The mean ± SEM for eight replicates is shown. E. Effect of eRF1 depletion on frameshifting with the native GGG intercodon and substituted stop codon (UGA or UAG). The mean ± SEM for a minimum of 10 replicates from at least two independent experiments is shown. A dotted line indicates the level of −1 PRF in the absence of any shRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.g002" target="_blank">Fig. 2</a>). **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001, n.s., not significant.