Fig. 3.

SMURF2-dependent degradation of vesicular PTEN. PTEN co-immunoprecipitations (co-IPs) were performed on human umbilical vein endothelial cells (HUVECs) treated for 30 min with panel reactive antibody (PRA) sera and blotted as indicated (a). Rab5 co-IPs were performed on PRA-treated HUVECs transfected with control or SMURF2 siRNA (b). Western blot analysis of whole-cell lysates of HUVECs transiently transfected with SMURF2 WT, SMURF2 DN, control siRNA, and SMURF2 siRNA (c). HUVECs transfected with control, SMURF2, or ZFYVE21 siRNA were treated with PRA and Rab5 co-IPs were performed (d). Recombinant FLAG-tagged ZFYVE21 (1 μg) and/or recombinant SMURF2 (1 μg) protein was incubated for 30 min at room temperature and analyzed by western blot (e). In vitro kinase assays were performed using various E2 ubiquitin ligases with SMURF2 protein (1 μg) as the E3 ubiquitin ligase and PTEN (1 μg) as substrate (f). In vitro kinase assays were performed using isolated SMURF2 WT or SMURF2 DN as E3 ubiquitin ligases, UbcH5c as the E2 ubiquitin ligase, and PTEN as substrate as indicated (g). HUVECs were pretreated with MG132 for 4 h or chloroquine for 30 min prior to PRA treatment at the indicated times prior to Rab5 co-IP and western blot analysis (h). Western blot analyses were repeated two to four times. Confocal microscopic studies analyzed ≥4 cells per group and was repeated three times. In vitro kinase assays were repeated twice. Representative data shown