Fig. 3. Increased ribosome occupancy at the TC of in vitro translated mRNAs under decreased concentrations of the recycling factor ABCE1.

a Western blot analysis of ABCE1 depletion in HeLa lysates. Cell lysates equivalent to 4 × 10⁵ cells treated with a CTR or ABCE1 siRNAs were loaded on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for ABCE1 and beta-actin. b Toeprint analysis of ribosome occupancy at the TC under ABCE1 depletion was performed after in vitro translation of the 200 + pA construct for 50 min at 33 °C in lysates from cells treated with CTR or ABCE1 siRNAs. Sanger sequencing reactions were run in parallel (G, T, C, A) to locate the positions of the toeprints. The position of the TC and the toeprint band corresponding to the ribosomes at the TC (+18) are indicated. c Relative quantification of the translation-dependent +18 band under ABCE1 conditions of 4 independent toeprinting experiments, normalized to the translation-independent mRNA signals and to the values of the Ctr KD conditions. Mean values ± standard deviation are shown and the values of the individual experiments are depicted by dots (Unpaired, two-sided statistical t-test: Ctr/ABCE1 p value: 0,0014). Source data are provided as a Source Data File.