Fig. 6: NEDD4 ubiquitinates DKK1 to regulate protein levels.

A Western blot of an in vitro ubiquitination assay. HEK293T cells were transfected with DKK1-GFP, Ub-HA, and either no NEDD4, FLAG-NEDD4 CS (cysteine mutant) or FLAG-NEDD4 WT (wild-type). Immunoprecipitation for GFP demonstrates HA positivity with NEDD4 WT, indicating ubiquitination of DKK1, but minimal ubiquitination with NEDD4 CS or no NEDD4. Representative image from n = 3 independent experiments. B Ubiquitination assay as in (A), but with DKK2-GFP construct. Minimal ubiquitination is observed in all conditions. Representative image from n = 3 independent experiments. C Western blot of HEK293T cells transfected with DKK1-Myc and FLAG-NEDD4 as indicated. Representative image from n = 5 biological experimental replicates. Quantitation of integrated density normalised to Actin shows that steady-state DKK1 protein levels are reduced with NEDD4 WT compared to either no NEDD4 or NEDD4 CS. ns not significant; **¹ P = 0.0012; **² P = 0.0061 (unpaired t test, two-tailed). D HeLa cells transfected with DKK1-GFP and FLAG-NEDD4 WT or CS, immunostained for FLAG and DKK1. Representative images from n = 3 independent experiments. Mean fluorescence intensity of DKK1 and NEDD4 quantified for individual cells and plotted graphically shows a negative linear correlation between DKK1 and NEDD4 WT levels, but not NEDD4 CS (simple linear regression). For transfection control for DKK1 construct, see Supplementary Fig. 16. E HeLa cells transfected with GFP and FLAG-NEDD4 WT or CS, immunostained for FLAG and GFP. Representative images from n = 3 independent experiments. Quantification of mean fluorescence intensity reveals no correlation between NEDD4 and GFP levels (Simple linear regression). F Sagittal sections through the second heart field (SHF) region of E9.5 wild-type and Wnt1-Cre; Nedd4^fl/fl embryos immunostained for NEDD4, DKK1, and GM130. Representative images from n = 5 independent embryos per genotype. Arrowheads indicate areas of co-localisation of NEDD4 with DKK1, in the neural crest cells (NCCs), which overlap with areas of the Golgi marker GM130. In Wnt1-Cre; Nedd4^fl/fl embryos, where NEDD4 is absent, DKK1 expression is increased. Source data are provided as a Source Data file.