Fig. 5: Both N- and C-terminal domains are needed for translational inhibition.

A–C Native gel shift assay with fluorescence labeled-Pdcd4 (green), either using the full-length Pdcd4, Pdcd4-NTD (residues 1-156), or Pdcd4-CTD (residues 157-469), for different 40S ribosomal subunit complexes (stained with ethidium bromide, shown in red). Both FL-Pdcd4 and FL-Pdcd4-NTD tightly co-migrate with the 40S (lane 2), and weakly with 43S (lane 3, <20% binding compared to lane 2). Only FL-Pdcd4 shows tight binding to the 43S in the presence of eIF4A (lane 4, see also Supplementary Fig. 9C). Note that FL-Pdcd4-NTD shows at least three extra non-ribosomal bands in lanes 3–4, which are not observed for FL-Pdcd4-NTD binding to 40S or eIFs independently, leaving their exact nature yet to be determined. FL-Pdcd4-CTD does not co-migrate with any ribosomal complexes, even in the presence of eIF4A. The presence of excess eIF4A causes tightening of the FL-Pdcd4-CTD band without appreciable shift (lane 4), most likely due to the interaction with free eIF4A outside the ribosome. Note the apparent overlap of FL-Pdcd4-CTD band with the 43S does not indicate they interact with each other, as they do not comigrate when the 43S band is shifted by further addition of eIF4F-mRNA (Supplementary Data Fig. 9B). D In vitro translation assay in HEK293T cells lysate using an mRNA encoding a firefly luciferase reporter sequence with β-globin 5′ UTR. The relative luciferase activity was measured in the presence of 250 nM Pdcd4 or its variant and normalized to a control performed without Pdcd4. ΔRibo mutant lacks the mRNA binding domain (residues 102–134) in the N-terminal domain. Error bars represent the mean ± SEM (n = 3). The experiment was repeated at least three times with similar results.