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. 2013 Feb 9;288(12):8289–8298. doi: 10.1074/jbc.M112.433185

FIGURE 1.

FIGURE 1.

NEDD4L interacts with Dvl2. A, the PY motif (PPXY) is highly conserved among three human Dvl proteins and the Drosophila Dishevelled protein. B, endogenous NEDD4L interacts with Dvl2. HEK293T cell lysates were subjected to immunoprecipitation (IP) with control IgG or anti-Dvl2 antibodies followed by anti-NEDD4L immunoblotting (IB). C, NEDD4L interacts with Dvl2 in vitro. GST pulldown assay using in vitro translated FLAG-Dvl2 and immobilized GST or GST-NEDD4L fusion protein is shown. Precipitated proteins were detected with anti-FLAG antibody. Loading of GST and GST-NEDD4L protein was shown by Coomassie staining. D, WW3 and WW2 domains of NEDD4L bind to Dvl2. After transfection with different GST-NEDD4L WW domains together with FLAG-Dvl2 for 48 h, HEK293T cells were harvested for GST pulldown followed by anti-FLAG immunoblotting. Total protein expression was confirmed by immunoblotting with whole cell lysates (WCL). E, the WW3 domain of NEDD4L is required for its interaction with Dvl2. After transfection with FLAG-NEDD4L CA or WW3 mutant together with HA-Dvl2 for 48 h, HEK293T cells were harvested for anti-HA immunoprecipitation followed by anti-FLAG immunoblotting. Total protein expression was confirmed by immunoblotting with whole cell lysates (WCL) (lower panels). F, the PY motif of Dvl2 is essential for its interaction with NEDD4L. The experiment was performed similarly as in D. G, the WW3 domain binds to wild-type, but not the AY mutant Dvl2. After transfection with GST-NEDD4L (WW3) together with wild-type or AY mutant FLAG-Dvl2 for 48 h, HEK293T cells were harvested for GST pulldown followed by anti-FLAG immunoblotting. Total protein expression was confirmed by immunoblotting with whole cell lysates. H, Cellular distribution of wild-type, CA mutant NEDD4L, wild-type and AY mutant Dvl2. HeLa cells transfected with wild-type or mutant GFP-NEDD4L or DsRed-Dvl2 individually were processed for immunofluorescence. Scale bar: 10 μm. I, wild-type, but not AY mutant Dvl2, recruits NEDD4L to puncta. HeLa cells transfected with wild-type or AY mutant DsRed-Dvl2 together with EGFP-NEDD4L CA were processed for immunofluorescence. Scale bar, 10 μm.