Figure 3
From: Inhibition of Wnt signalling by Notch via two distinct mechanisms
Receptor trafficking is required for ΔEGF_N1 to reduce β-catenin activity. (A) Membrane bound ΔEGF_N1 localised in the late endosome. Immunofluorescence analysis of ΔEGF_N1 and Rab7 proteins. Scale bar is 25 μm. (B) ΔEGF_N1 accumulated when receptor trafficking was blocked. Receptor trafficking was blocked by expressing a dominant negative form of Vps4. Western blot analysis of ΔEGF_N1 and DNVps4 showed more ΔEGF_N1 accumulation in cells when receptor trafficking was blocked. Expressed proteins were detected by probing the western blot with an antibody that recognises the myc epitope tag found within ΔEGF_N1 and an antibody that recognizes GFP. Tubulin is shown as a loading control. The position of molecular weight markers is shown in KDa. (C) ΔEGF_N1 accumulated at the cell surface when endocytosis was blocked. Immunofluorescence analysis for co-localization of ΔEGF_N1 with a DN form of Dynamin (K44ADyn2) protein. Scale bar is 25 μm. (D, E) Blocking endocytosis abrogated the ability of ΔEGF_N1 to inhibit S45Fβ-catenin-driven transcriptional activity. Endocytosis was blocked by expressing a dominant negative form of Dynamin (K44ADyn2) (D) or DN form of Rab5 (S34NRab5) (E). (F) Schematic showing the structure of the ΔEGF_N1, ΔEGF_N1 W1758A and ΔEGF_N1 R1994A constructs (G, H) ΔEGF_N1 requires Deltex to inhibit Wnt signalling. Luciferase assays indicated that ΔEGF_N1 R1994A (G) and Deltex (H) eliminated the ability of ΔEGF_N1 to inhibit the S45Fβ-catenin-driven transcriptional activity, while ΔEGF_N1 W1758A or Suppressor of Deltex (SuDx) had no significant effect. HEK293T cells were transfected with the Wnt reporter plasmid pTCF-AdTATA and Wnt signalling was activated by expressing S45Fβ-catenin. Experiments were performed in triplicate. pRL-CMV was used as a transfection control and cells were lysed 48 h post transfection to determine luciferase activity. Data are presented as mean fold change (± SEM) in RLU (NS P > 0.05; *P < 0.05, **P < 0.01, one-way ANOVA and Tukey’s post-hoc test, N = 3). Original uncropped Western Blot images of panel B are shown in Supplementary Fig. 5
