Unless otherwise stated, mRNAs were translated in FlexiH rabbit reticulocyte lysate (FlexiHRRL, Promega) programmed with template mRNA at 50 mg/ml. Typical reactions were of 10 ml and composed of 60% (v/v) FlexiHRRL, 20 mM amino acids (lacking methionine), 500 mM MgOAc, 2 mM DTT, 5U RNase inhibitor (RNAguard, GE Healthcare Life Sciences), 130 mM-160 mM KCl (optimised for each batch of FlexiHRRL) and 0.2 MBq [ 35 S]methionine. Reactions were incubated for 1 h at 30uC and stopped by the addition of an equal volume of 10 mM EDTA, 100 mg/ml RNase A followed by incubation at room temperature for 20 minutes. Samples were prepared for SDS-PAGE by the addition of 4 volumes of 4X Laemmli's sample buffer, boiled for 3 minutes and resolved on 12% SDS-PAGE gels. The relative abundance of products on the gels was determined by direct measurement of [ 35 S]-methionine incorporation using a Packard Instant Imager 2024. eIF4G-depleted RRL was prepared and used as described previously [26] .