Supplementary Figure 5: OTUD3 is a more potent regulator of PTEN than USP13, and HAUSP has no significant effect on PTEN protein level.

a, Increasing amounts of Flag-USP13 were transfected into MCF7 (left) and HEK293T (right) cells. After 48 h, PTEN protein levels were detected. b, Co-immunoprecipitation of endogenous PTEN, USP13 and OTUD3 in MCF7 cells. Protein extracts were immunoprecipitated with antibody against PTEN, USP13 or OTUD3, followed by immunoblotting with antibodies indicated. c, Increasing amounts of shRNA-USP13 were transfected into MCF7 cells. After 72 h, PTEN protein levels were detected. d, Protein level analysis of PTEN in the presence of overexpressed USP13 or OTUD3. MCF7 cells were overexpressed with Myc-USP13 or Flag-OTUD3. e, Protein level analysis of PTEN in MCF7 cells stably expressing the indicated shRNA. f, MCF7 cells were transfected with a constant amount of shRNA-USP13 and increasing amounts of Myc-OTUD3 (left), or a constant amount of shRNA-OTUD3 and increasing amounts of Myc-USP13 (right). After 48 h, PTEN protein levels were detected. g, HCT116 cells transfected with the indicated constructs were treated with MG132 for 8 h before harvest. PTEN was immunoprecipitated with anti-PTEN and immunoblotted with anti-HA. h, siRNA-resistant OTUD3 was introduced into the HCT116 cells together with the OTUD3 siRNA. PTEN ubiquitination was measured. i, The PTEN ubiquitination linkage was analysed in HCT116 cells transfected with OTUD3and the indicated ubiquitin WT or K48-only plasmids. j, Increasing amounts of HAUSP plasmids were transfected into the MCF7 cells and the lysates were subjected to western blot analysis. k, Knockdown of HAUSP by individual siRNAs had no significant effect on PTEN protein level but downregulated MDM2 protein level (positive) in MCF7 cells. l, Effectiveness of HAUSP knockdown by individual siRNAs was measured by quantitative RT-PCR. Data are shown as mean ± s.d. n = 3 independent experiments. (**, P < 0.01, Student’s t-test). All panels are representative results of three or more independent experiments. Statistics source data can be found in Supplementary Table 4. Uncropped images of blots are shown in Supplementary Fig. 9.