Xenbase Image ID: 78983

Fig. 6. Dishevelled DIX and PDZ domains are required for inhibition of RBPJκ. (A) Schematic of the structure of Dishevelled and the deletion constructs used. (B) The C terminus of mDvl2 is not required for inhibition of RBPJκ activity. CHO-K1 cells were transfected with RBPJκ-Luc and pRL-CMV, and vectors encoding δN-mN1 and the mDvl2 constructs illustrated. Data are presented as mean fold change (±s.e.m.) in RLU relative to δN-mN1 alone. Both δC-mDvl2 and mDvl2, but not δN-mDvl2, inhibit Notch signalling (one-way ANOVA and Tukey's post-hoc test, n≥3). (C-E) XDvl2 but not Ds1 inhibits endogenous Notch signalling in vivo. Xenopus laevis embryos were injected in one blastomere of a two-cell embryo with mRNA encoding β-gal and XDvl2 (C,C′) or β-gal and Ds1 (D,D′). Ciliated cell precursors were detected by α-tubulin expression and the injected side was determined by X-Gal staining. Images of the uninjected (UI) and injected sides of the same embryo are shown. (E) Ciliated cell precursors were quantified as in Fig. 2D. Data are presented as the mean number of precursors counted (±s.e.m.). XDvl2 significantly increased ciliated cell precursor specification but Ds1 did not (two-tailed paired t-test). XDvl2 also increased precursor specification compared with Ds1 (two-way ANOVA and Bonferroni's post-hoc test). (F) Ds1 does not inhibit XSu(H) activity. CHO-K1 cells were transfected with NRE Su(H)-Luc and pRL-CMV, and vectors encoding XSu(H)-ANK, XDvl2 or Ds1. Data are presented as mean fold change (±s.e.m.) in RLU relative to XSu(H)-ANK alone (one-way ANOVA and Tukey's post-hoc test, n=3). (G) Ds1 shows greatly reduced XSu(H) binding. CHO-K1 cells expressing XSu(H)-ANK-myc and XDvl2-GFP or Ds1-GFP were subjected to immunoprecipitation using GFP-Trap beads. Immunoprecipitation samples were analysed by immunoblotting for myc and GFP, alongside total lysates. Positions of molecular weight markers (in kDa) are shown. Scale bar: 500 μm. **P<0.01; ***P<0.001; ns, P>0.05. Image published in: Collu GM et al. (2012) © 2012. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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