Fig 1: DDX6 interacts with ribosomal proteins in human cells.(A) The interaction between the recombinant NusA-Strep-DDX6 and purified human ribosomal proteins was analyzed by SDS-PAGE and stained with Coomassie blue. Input lysate (1%) and bound fractions (20%) were loaded. (B) Western blot showing the interaction between GFP-tagged DDX6 full-length/N-ter/C-ter with HA-tagged RPL22 and endogenous RPS3A in human HEK293T cells. GFP-tagged MBP served as a negative control. For the GFP-tagged proteins, the HA-tagged RPL22, and the endogenous RPS3A, 1% of the input and 20% of the immunoprecipitate were loaded. N-ter: N-terminus; C-ter: C-terminus. (C) Immunoprecipitation assay showing the interaction of GFP-tagged DDX6 (wild-type or the indicated mutants) with HA-tagged RPL22 or endogenous CNOT1 in HEK293T cells. Samples were analyzed as described in B. (D) DDX6 KO HEK293T cells were transfected with the control Renilla luciferase (R-LUC) reporter or a reporter containing 30 x rare codons and GFP-tagged DDX6 wild-type or mutants. After treating cells with ActD for 8 hr R-LUC mRNA levels were analyzed by northern blotting. 18 S rRNA ethidium bromide staining shows equal loading. (E) Relative control reporter mRNA levels from panel D were defined as 100%. Relative 30 x rare codon reporter mRNA levels were plotted. Bars represent the mean value and error bars the standard deviation (n=3). (F) Immunoblot illustrating the expression of proteins used in the assay shown in panel D. Figure 2—source data 1.Original file for the SDS-PAGE in Figure 2A. Figure 2—source data 2.PDF containing Figure 2A and original scans of the relevant SDS-PAGE with highlighted bands and sample labels. Figure 2—source data 3.Original file for the western blot in Figure 2B. Figure 2—source data 4.PDF containing Figure 2B and original scans of the relevant western blot analysis with highlighted bands and sample labels. Figure 2—source data 5.Original file for the western blot in Figure 2C. Figure 2—source data 6.PDF containing Figure 2C and original scans of the relevant western blot analysis with highlighted bands and sample labels. Figure 2—source data 7.Original file for the northern blot in Figure 2D. Figure 2—source data 8.PDF containing Figure 2D and original scans of the relevant northern blot analysis with highlighted bands and sample labels. Figure 2—source data 9.Original file for the western blot in Figure 2F. Figure 2—source data 10.PDF containing Figure 2F and original scans of the relevant western blot analysis with highlighted bands and sample labels.