Journal: Science Advances

Article Title: A Wnt-induced conformational phospho-switch in DVL3 controls association with Frizzled receptors and Wnt/β-catenin signaling

doi: 10.1126/sciadv.aed8899

Figure Lengend Snippet: ( A ) Schematic representation of the proximity-labeling experimental workflow. Expression of WT and phospho-switch mutant proteins fused to a promiscuous biotin ligase (TurboID) was induced by doxycycline. Biotinylated proteins were identified by LC-MS/MS. Created in BioRender. Gybel, T. (2026) https://BioRender.com/pyxzao0 . ( B ) Volcano plots comparing proteins enriched (right) or depleted (left) in the interactomes of phospho-switch mutants in comparison to WT. ( C ) The potency of each interaction was calculated as the Euclidean distance from the origin of the volcano plots comparing the interactome of phospho-switch mutants with WT. The corresponding values were plotted as a function of the net charge (at pH 7.2) proximal to the DEP domain and fitted using a sigmoidal function to determine the charge threshold for each FZD. Axin interaction with DVL3 did not show significant difference between the phospho-switch mutants and WT. ( D ) FZD-DEP interface, pIDR2-DEP interface, and the two overlapping epitopes mapped onto the FZD-DEP structure (PDB ID: 8WMA). ( E ) Mechanistic model of DVL phospho-switch in Wnt/β-catenin signaling. Wnt stimulus results in FZD clustering, stabilization of DVL at the membrane, and more efficient CK1δ/ε-mediated DVL phosphorylation that leads to the accumulation of negative charge proximal to the DEP domain. Charge-mediated DVL intramolecular interaction competes with FZD binding, and when the charge threshold is reached, DVL dissociates from FZD (step 1). This event must be accompanied by the activation of LRP5/6 coreceptors (step 2), to propagate Wnt signal to downstream components. Created in BioRender. Gybel, T. (2026) https://BioRender.com/tremyul .

Article Snippet: The expression of DVL was induced by doxycycline (1 μg/ml; HY-N0565B, MedChemExpress) treatment for 24 hours and supplemented with 50 μM biotin (SC204706A, Santa Cruz Biotechnology) afterward.

Techniques: Labeling, Expressing, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Comparison, Membrane, Phospho-proteomics, Binding Assay, Activation Assay

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Levels of the 7TGP fluorescent Wnt reporter in HEK293T cells transfected with control siRNA, Dvl1-specific siRNA, Nup358-specific siRNA, or a combination of Nup358-specific siRNA and Dvl1-specific siRNA and treated with either Wnt3a or vehicle. b. Western blot analysis and relative quantification of protein levels of Dvl1 and Axin1 in HEK293T cells transfected with either control or Nup358-specific siRNA. α-Tubulin and GAPDH were used as loading controls. Data are expressed as mean ± SD. **p ≤ 0.01. c. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with either control or Nup358-specific siRNA. d. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with control or Nup358-specific siRNA and treated with vehicle or 1,6-hexanediol 5% for 2 minutes. e. Fluorescence recovery after photobleaching (FRAP) and fusion analysis of Dvl1 condensates in HEK293T cells transfected with Nup358-specific siRNA and EGFP-Dvl1.

Article Snippet: Precleared protein solution was incubated over-night at 4°C on a rotator with either a specific anti-Dvl1 antibody (Santa Cruz #8025) at the final dilution of 1:400 or with the same amount of control rabbit IgG.

Techniques: Transfection, Control, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. HEK293T cells were transfected with β-catenin-GFP plasmid and control or Nup358-specific siRNAs and GFP expression was analyzed by confocal microscopy. The percentage of GFP-positive cells and GFP intensity per cell were quantified. Data are expressed as mean ± SD. ***p ≤ 0.001. b. Immunofluorescence staining for Dvl1 and ADP-ribosylation in HEK293T cells transfected with either control or Nup358-specific siRNAs. c. Western blot analysis of co-immunoprecipitation of ADP-ribosylation and Axin1 in HEK293T cells transfected with control or Nup358-specific siRNA. Axin1 levels in immunoprecipitates were quantified relative to controls and normalized to Axin1 input levels. Data are expressed as mean ± SD. **p ≤ 0.01 d. Left: Western blot analyses of Nup358 and Axin1 in HEK293T cells transfected with control or Nup358-specific siRNA and treated with 200 nM XAV-939 or vehicle for 72 hours. α-Tubulin was used as loading control. Right: Quantification of Axin1 protein levels (n=2 experiments). e. Quantification of fluorescent Wnt reporter activity by flow cytometry analysis in HEK293T cells transfected with control siRNA or Nup358-specific siRNA and treated with 200nM XAV-939 or vehicle for 72 hours. Data are expressed as mean ± SD. **p ≤ 0.01 and ***p ≤ 0.001.

Article Snippet: Precleared protein solution was incubated over-night at 4°C on a rotator with either a specific anti-Dvl1 antibody (Santa Cruz #8025) at the final dilution of 1:400 or with the same amount of control rabbit IgG.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Confocal Microscopy, Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Activity Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Schematic representing different functional domains of Nup358. b. Fluorescent Wnt reporter activity in HEK293T cells transfected with control siRNA, Nup358-specific siRNA, Ubc9-specific siRNA, or treated with the SUMOylation inhibitor 2-D08. c. Schematic representing different HA-tagged truncated forms of Nup358 that were transiently expressed in HEK293T cells. d. Fluorescent Wnt reporter activity in HEK293T cells transfected with either control or Nup358-specific siRNA alone or in combination with different HA-tagged truncated forms of Nup358 were analyzed by confocal microscopy (top) and quantified (bottom). Data are expressed as mean ± SD. ****p ≤ 0.0001 e. Western blot analysis of co-immunoprecipitation of HA-Nup358 and EGFP-Dvl1 transiently expressed in HEK293T cells. GAPDH was used as loading control. f. Immunofluorescence of HEK293T cells transfected with HA-Tagged Nup358 1–1133 and EGFP-Dvl1and stained with an anti-HA antibody. g. Immunofluorescence staining for endogenous Dvl1 and Nup358 in HEK293T cells transfected with either scramble control or Nup358-specific siRNA alone or in combination with HA-Tagged Nup358 1–1133 fragment were analyzed by confocal microscopy (top) and quantified (bottom). Data are expressed as mean ± SD. **p ≤ 0.01.

Article Snippet: Precleared protein solution was incubated over-night at 4°C on a rotator with either a specific anti-Dvl1 antibody (Santa Cruz #8025) at the final dilution of 1:400 or with the same amount of control rabbit IgG.

Techniques: Functional Assay, Activity Assay, Transfection, Control, Confocal Microscopy, Western Blot, Immunoprecipitation, Immunofluorescence, Staining