FIGURE 4.

K270 mutation prevents ubiquitination of Arrdc4 and inhibits Arrdc4‐mediated EV biogenesis. (a, b) Representative immunoblots of ubiquitination assay performed in HEK293T cells by expressing GFP‐tagged Arrdc4 (WT/K270R), Flag‐tagged Nedd4, Nedd4‐2 and Smurf1 (WT and catalytically inactive mutant, CM) and HA‐tagged ubiquitin. Arrdc4 ubiquitination by Nedd4, Nedd4‐2 and Smurf1 is decreased by mutation at K270. (c–e) Release of extracellular vesicles (EV_100‐200nm normalised to equal number of cells) by Arrdc4^WT is significantly increased in presence of Nedd4‐2 and Smurf1, but not Nedd4, and this is inhibited by the Arrdc4^K270R mutant. +, data compared with control whereas * indicates significance when compared with Arrdc4^WT. ^+/*P < 0.05, ^++/**P < 0.01, ns = not significant