Extended Data Fig. 9: Recombinant Dap1b binds to the polypeptide exit tunnel of rabbit ribosomes.
From: A molecular network of conserved factors keeps ribosomes dormant in the egg
a-b, Western blot of the total in vitro translation reaction shown in Fig. 4c (a) and Fig. 4e (b). Uncropped images of membranes are provided in Supplementary Fig. 2a, b. c, Translation activity assays (Fig. 4a top) of renilla luciferase mRNA upon addition of increasing concentrations of C-terminal Dap and Dap1b peptides. BSA and N-terminal Bac7 are used as negative and positive controls, respectively (n = 4 biologically independent samples). Dots represent means and error bars are standard deviation (SD). d, Ribosome binding assays (Fig. 4a bottom) of in vitro translated FLAG-tagged Dap-Dap1b chimeras compared to full-length (WT) Dap and Dap1b. A representative Western blot from a single experiment is shown on the left; quantification of three independent experiments is shown on the right. Data are represented as scatter dot plots with means ± standard deviation (SD). Significance was assessed with Kruskal-Wallis followed by Dunn’s two-sided test. Uncropped images of membranes are provided in Supplementary Fig. 2d. e, Mass spectrometry data of ribosomes isolated from habp4−/− and WT (left), and from dap−/− dap1b−/− and WT (right) embryos at 1 hpf, represented as volcano plots (n = 3 independent experiments). Permutation-based false discovery rates (FDRs) are displayed as dotted (FDR < 0.01) and dashed (FDR < 0.05) lines.
