Fig 1: NEDD4 and NEDD4L selectively degrade Lgr4 and Lgr5 but not FZD receptors of Wnt signalling pathway AHEK293T cells were transfected with empty vector (EV), LGR4-Flag, MYC-NEDD4 WT or C854S (CS) mutant, MYC-NEDD4L wild-type (WT) or C962A (CA) mutant, followed by Western blot analysis of the indicated antibodies.B, CHEK293T cells were transfected with V5-FZD4 (B) or V5-FZD5 (C) with or without MYC-NEDD4 or MYC-NEDD4L or empty vector (EV) as control. Lysates were subjected to Western blotting using the indicated antibodies.DSubcellular localisation of SNAP-FZD5 in HEK293T cells co-expressed with the indicated plasmids. Surface SNAP-FZD5 was labelled with SNAP Alexa-488 for 10 min. Scale bars, 10 µm.EQuantitation of fluorescent intensity in total and surface LGR5 and FZD5 with the indicated transfections of NEDD4 and NEDD4L. Data are presented as percentage of fluorescence intensity compared to the EV control in triplicate for LGR5 and duplicate for FZD5. Error bars represent ± SEM. P-values were determined using the unpaired two-sided t-test (*P < 0.05; ***P < 0.001).FHEK293T cells WT, NEDD4 CRISPR or NEDD4L CRISPR mutants were transfected with LGR5-Flag. Twenty-four hours later, cells were treated with cycloheximide (Chx) (50 µg/µl) and were collected at different time points as indicated. Cell lysates were subjected to Western blot analysis using the indicated antibodies. Quantitation of the blots is shown at the bottom. Data represent mean ± standard error of at least three independent experiments. P-values were determined using the unpaired two-sided t-test compared between Nedd4 and WT (indicated by *) or Nedd4l and WT (indicated by #) at the same time point *P < 0.05; ## P < 0.01).G, HHEK293T cells were transfected with constructs expressing LGR5-Flag, HA-Ubiquitin, EV or the indicated NEDD4 (G) or NEDD4L (H) plasmids. Cells were treated with Bafilomycin A1 followed by anti-Flag IP and Western blot analysis using the indicated antibodies.