Supplementary Figure 4: OTUD3 stabilizes PTEN protein.

a, Protein level analysis of PTEN in the presence or absence of overexpressed DUBs. HCT116 colon cancer cells were overexpressed with each of the Flag-tagged DUBs. b, Quantification of the PTEN protein levels relative to β-actin for Fig. 1b. n = 3 independent experiments (**, P < 0.01, Student’s t-test). c, Increasing amounts of plasmids encoding other members of OTU sub-family of DUBs were each transfected into HCT116 cells. The endogenous PTEN protein levels were examined by western blot. d, Knockdown of OTUD3 by pool siRNAs (left) or individual siRNAs (middle) or overexpression of OTUD3 (right) had no significant effect on PTEN mRNA level in MCF7 cells. n = 3 independent experiments (NS: no significance, Student’s t-test). e, MCF7 cells stably expressing the indicated shRNA were treated with cycloheximide (10 μg ml⁻¹), and harvested at the indicated times. The left panels show immunoblots of PTEN and OTUD7A. Right panel: quantification of the PTEN protein levels relative to β-actin. n = 3 independent experiments (NS: no significance, two-way ANOVA test). f, Domain structure of OTUD3 and OTUD7A. g, Co-immunoprecipitation assays in four types of cancer cell lines to detect the endogenous interactions. The total cell lysates were immunoprecipitated with anti-OTUD3, anti-PTEN or control IgG as indicated. Both the lysates and the immunoprecipitates were subjected to western blot analysis. h, GST pull-down assays were performed to map the domain of OTUD3 required for interaction with PTEN. i, GST pull-down assays were performed to map the domain of PTEN required for interaction with OTUD3. 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.