Fig. 7

WWP2 reduces the protein level of DDX3X by promoting its K63-linked polyubiquitination and proteasomal degradation. A, B Representative Western blots of WWP2 and DDX3X protein levels. HUVECs were transfected with expression plasmids encoding HA-WWP2 (A) or three siRNA sequences of WWP2 (B). C–F Representative Western blotting analysis of WWP2 and DDX3X protein levels (C, E) and quantitative analysis of DDX3X protein expression levels (D, F). HUVECs were transfected with expression plasmid encoding HA-WWP2 (C, D) or WWP2 siRNA (E, F) and then treated with CHX (20 μM) for the indicated time periods. Values are shown as mean ± SD (***P < 0.001, two-way ANOVA with Bonferroni’s multiple comparison post hoc test). G, H Co-immunoprecipitation analysis was performed to assess the role of WWP2 in regulating the levels of DDX3X ubiquitination. HUVECs were transfected with HA-WWP2 plasmid (G) or WWP2 siRNA (H), and the levels of DDX3X ubiquitylation were analyzed by immunoprecipitation of DDX3X followed by anti-HA or anti-Ub immunoblotting. I Effects of the indicated K-only ubiquitin mutants (K48 and K63) on WWP2-mediated DDX3X polyubiquitination. HUVECs were transfected with the indicated constructs. J Effects of lysosome inhibitor CQ and proteasome inhibitor MG132 on WWP2-mediated K63-linked polyubiquitination of DDX3X. HUVECs were transfected with expression plasmids encoding HA-WWP2 and Ub K63, and then treated with 50 μM CQ for 24 h or 10 μM MG132 for 6 h, or DMSO as control. K, L Representative Western blotting analysis of HA-WWP2 and DDX3X protein levels (K) and quantitative analysis of DDX3X protein expression levels (L). Values are shown as mean ± SD (***P < 0.001, NS = not significant, two-way ANOVA with Bonferroni’s multiple comparison post hoc test). CHX, cycloheximide; CQ, chloroquine; HUVECs, Human umbilical vein endothelial cells; NC, negative control; siRNA, small interfering RNA; Ub, ubiquitin