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. 2016 Mar 24;35(11):1186–1203. doi: 10.15252/embj.201592901

Figure 8. DDX6 requires interaction with NOT1 and the integrity of the FDF‐binding surface to mediate silencing.

Figure 8

  • A–D
    Immunoprecipitation assay showing the interaction of GFP‐tagged DDX6 (wild type or the indicated mutants) with HA‐tagged decapping factors and 4E‐T in HEK293T cells. Inputs (1% for the HA‐tagged proteins and 3.5% for the GFP‐tagged proteins) and immunoprecipitates (20 and 7%, respectively) were analyzed by Western blotting using anti‐HA and anti‐GFP antibodies. GFP‐MBP served as a negative control.
  • E
    Immunoprecipitation assay showing the interaction of GFP‐tagged NOT1 with HA‐tagged DDX6 (wild type or the indicated mutants) in HEK293T cells. Samples were analyzed as described in (A–D).
  • F
    Control HeLa cells (transfected with a scrambled shRNA) or cells depleted of DDX6 were transfected with a mixture of three plasmids: the R‐Luc‐8xlet‐7‐poly(A) or the corresponding reporter carrying mutations in let‐7‐binding sites (R‐Luc‐Mut, Mut), a plasmid expressing F‐Luc as a transfection control, and a plasmid expressing shRNA‐resistant versions of GFP–DDX6 (wild type or the indicated mutants) or MBP. For each condition, Renilla luciferase activity was measured, normalized to that of the F‐Luc transfection control and set at 100% in cells expressing R‐Luc‐Mut. The left panel shows the normalized Renilla luciferase activities in control cells (i.e. cells treated with scrambled shRNA and expressing MBP). The right panel shows relative fold derepression for each condition for the R‐Luc‐8xlet‐7‐poly(A) after normalization. Mean values ± standard deviations from 4 independent experiments are shown.
  • G
    Western blot showing the efficiency of the DDX6 knockdown. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. PABP served as a loading control.
  • H
    Control HeLa cells (transfected with a scrambled shRNA) or cells depleted of 4E‐T were transfected with a mixture of three plasmids as described in panel (F). The left panel shows normalized Renilla luciferase activities in control cells (i.e. cells treated with scrambled shRNA). The right panel shows the relative fold derepression for the R‐Luc‐8xlet‐7‐poly(A) (after normalization) in 4E‐T‐depleted cells relative to control cells. Mean values ± standard deviations from three independent experiments are shown.
  • I
    Western blot showing the efficiency of the 4E‐T knockdown. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. Tubulin served as a loading control.

Source data are available online for this figure.