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. 2016 Mar 11;35(9):974–990. doi: 10.15252/embj.201593634

Figure 8. The NED and NIM are functionally equivalent.

Figure 8

  1. Tethering assay using a chimeric Nanos protein and the F‐Luc‐5BoxB reporter in S2 cells. The chimeric Nanos protein contains the NIM of human Nanos2 (either wild type (NIM‐ZnF) or mutated (NIM*‐ZnF)), fused to the Dm ZnF domain. A plasmid expressing GST served as a negative control. F‐Luc activity and mRNA levels were analyzed as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments.
  2. Northern blot of representative RNA samples corresponding to the experiment shown in (A).
  3. The activity of GST‐HA‐tagged Nanos chimeric protein was tested in S2 cells expressing an F‐Luc‐hb reporter. A plasmid expressing GST served as a negative control. F‐Luc activity and mRNA levels were analyzed as described in Fig 1B. The panel shows mean values ± standard deviations from three independent experiments.
  4. Northern blot of representative RNA samples corresponding to the experiment shown in (C).
  5. Western blot analysis showing the interaction of the HA‐tagged Nanos chimeric protein (NIM‐ZnF or NIM*‐ZnF) with endogenous Dm NOT1 and NOT3. HA‐MBP served as a negative control.
  6. Tethering assays in human HEK293T cells, using a β‐globin reporter containing 6 binding sites (6xbs) for the MS2 protein and MS2‐HA‐tagged Dm Nanos or NED fragment. A plasmid expressing an mRNA lacking MS2 binding sites (Control) served as a transfection control. The β‐globin‐6xbs mRNA levels were normalized to those of the control mRNA and set to 100 in the presence of MS2‐HA. The panel shows mean values ± standard deviations from three independent experiments.
  7. Northern blot of representative RNA samples corresponding to the experiment shown in (F).
  8. Co‐immunoprecipitation assay in human HEK293T cells showing the interaction of the HA‐tagged Dm Nanos and the NED fragment with GFP‐tagged NOT3 in human cells. HA‐MBP served as a negative control. The inputs (1%) and bound fractions (10% of HA‐tagged proteins and 30% of GFP‐NOT3) were analyzed by Western blotting.

Source data are available online for this figure.