Fig. 8: BioE3 using bio^GEFUb^WT identifies targets of activated NEDD4.

a–c Ubnc (L73P) mutation impairs NEDD4~Ub transthiolation and autoubiquitination. a Western blot (upper) and Coomassie staining of NEDD4 transthiolation assay, using Ub^WT loaded Ube2D3 (E2~Ub^WT) or Ubnc loaded Ube2D3 (E2 ~ Ub^L73P). b Coomassie staining showing that Ub^WT and Ubnc were at similar levels in the reaction. c Western blot (upper) and Coomassie staining of NEDD4 autoubiquitination assay using purified Ube1 (E1), Ube2D3 (E2), Ub^WT or Ubnc together with NEDD4^WT or NEDD4^3M (E3s). Ubiquitination reactions were stopped at indicated time-points. NEDD4^WT and NEDD4^3M autoubiquitination is impaired by L73P mutation on Ub (black arrowhead). Data are representative of 2 independent experiments with similar results. d, e Western blot of BioE3 experiments performed on HEK293FT stable cell line expressing TRIPZ-bio^GEFUb^WT transiently transfected with d EFS-BirA-NEDD4^WT, NEDD4^CA, NEDD4^ΔC2 or NEDD4^ΔC2,CA and e EFS-BirA-NEDD4^3M or EFS-BirA-NEDD4^3M,CA. Active, auto-ubiquitinated and biotinylated BirA-NEDD4^ΔC2 and BirA-NEDD4^3M are depicted with black arrowheads and ubiquitinated substrates with bars. Dots indicate endogenous biotinylated carboxylases. Cells in (e) were also treated with the DUB inhibitor PR619. f Confocal microscopy of BioE3 experiment performed on U2OS stable cell line for TRIPZ-bio^GEFUb^WT transfected with EFS-BirA-NEDD4^WT, BirA-NEDD4^ΔC2 or BirA-NEDD4^3M. Biotinylated material is stained with fluorescent streptavidin (Strep, magenta), and BirA with specific antibody (green). Black and white panels show the green and magenta channels individually. Scale bar: 8 μm. g Volcano plot of LC-MS analysis comparing streptavidin pull-downs of BioE3 experiments performed on HEK293FT stable cell line expressing TRIPZ-bio^GEFUb^WT and transfected with EFS-BirA-NEDD4^3M or BirA-NEDD4^3M,CA (n = 3 biological replicates). Proteins significantly enriched (Log2 NEDD4^3M/NEDD4^3M,CA > 0 and p-value < 0.05) were considered as NEDD4 targets. Statistical analyses were performed by two-sided Student’s t-test. Data are provided as Supplementary Data 7. h Western blot validations of NEDD4 targets identified in (g): CCT8 and TP53BP2. Arrowheads and bars point to unmodified and Ub modified proteins, respectively. IN: input; St PD: Streptavidin pull-down. a–h Molecular weight markers are shown to the left of the blots in kDa. d–h Data are representative of 3 independent transfection experiments with similar results. Source data are provided in the Source data file.