Figure 5.

OTUB2 interacts with and stabilizes YAP. (A) Molecular docking assays of OTUB2 and YAP. (B) VSMC lysates were immunoprecipitated with an anti-YAP antibody, and HEK293T cells transfected with Flag-tagged OTUB2 and HA-tagged YAP were subjected to Co-IP assays. (C) Immunofluorescence staining were performed using antibodies against OTUB2 (red) and YAP (green). DAPI (blue). Scale bars, 50 μm. (D) SUMOylation levels of OTUB2 in VSMCs treated with GM or CM. (E-F) Western blot analysis and quantification of YAP expression. n = 3 per group. (G) Control or OTUB2-silenced VSMCs were treated with cycloheximide (CHX) and subjected to Western blot. (H) Control or OTUB2-overexpressing VSMCs were treated with CHX and subjected to Western blot. (I) An empty vector and Flag-OTUB2 C51S were transfected into VSMCs, which were then treated with CHX, and a quantitative analysis of the half-life of the YAP protein was performed. (J) Schematic diagram showing the wild-type and truncated YAP and OTUB2 constructs. (K) Representative immunoblots showing the interaction between OTUB2 and WT or truncated YAP, as indicated, as assessed by IP. (L) Representative immunoblots showing the interaction between YAP and WT or truncated OTUB2, as indicated, as assessed by IP. (M) K48-linked ubiquitination of YAP was measured by immunoblotting after OTUB2 was silenced. (N) K48-linked ubiquitination of YAP was measured by immunoblotting after OTUB2 was overexpressed. (O) K63-linked ubiquitination of YAP was measured by immunoblotting. (P) An empty vector, Flag-OTUB2 and Flag-OTUB2 C51S mutants were transfected into VSMCs. The K48-linked ubiquitination of YAP was measured by immunoblotting. Statistical significance was assessed using one-way ANOVA followed by Dunnett's test (E, F). All values are presented as mean ± SD. *P < 0.05.