Figure 4
From: PTEN stabilizes TOP2A and regulates the DNA decatenation
PTEN maintains TOP2A stability by promoting OTUD3 catalyzed TOP2A deubiquitination.
(a) Western blot analysis of Top2a and Pten. MEF Pten+/+ or Pten−/− cells were treated with MG132 prior to western blot analysis with indicated antibodies. (b) Western blot analysis of Top2a and Pten. MEF Pten+/+ or Pten−/− cells were treated with MG132 prior to CHX treatment for indicated time periods followed by western blot analysis of Top2a and Pten levels. (c) Ubiquitination assays. HCT116 PTEN+/+ or PTEN−/− cells and HCT116 PTEN−/− cells ectopically expressing Flag-tagged PTEN or PTEN C124S were transfected with His-ubiquitin and treated with MG132 prior to His-tagged pull-down and western blot analysis of ubiquitinated-TOP2A with antibody to TOP2A. Non-specific bands are shown as loading control. (d) Co-immunoprecipitation assays. PTEN antibody or IgG was used for immunoprecipitation in HCT116 cell extracts prior to western blotting of PTEN and OTUD3. (e, f) Co-immunoprecipitation assays. MEFs, HCT116 PTEN+/+ or HCT116 PTEN−/− cell extracts were immunoprecipitated with OTUD3 antibody or mouse IgG. Western blot was then used to evaluate for presence of TOP2A and OTUD3. (g) OTUD3 and TOP2A in vitro binding assay. SA-tagged OTUD3 and flag-tagged TOP2A-N and TOP2A-C were purified and incubated together before flag pull-down. Note that the C-terminus of TOP2A is able to bind OTUD3 in vitro. (h) Ubiquitination assays. HCT116 cells were transfected with either empty vector or HA-OTUD3 together with His-ubiquitin prior to MG132 treatment. Ubiquitinated proteins were enriched with His pull-down and blotted with TOP2A antibody. Non-specific bands are shown as loading control. (i) Western blot analysis of TOP2A, PTEN and overexpressed HA-OTUD3. HA-tagged OTUD3 was overexpressed in HCT116 PTEN+/+ and PTEN−/− cells before western blotting with indicated antibodies.
