Figure 1.
(A) OTUD5 interacts with p53 in yeast two-hybrid assay. AH109 yeast cells were cotransformed with indicated plasmids, then plated on SD/–Ade/–His/–Leu/–Trp selection mediums. (B) The HEK293 cells were cotransfected with Flag- OTUD5 and Myc-p53, and cell lysates were immunoprecipitated with indicated antibodies and immunoblotted with anti-Myc or anti-Flag antibody. (C) Immunoprecipitation of p53 from U2OS cells, followed by western blotting of the precipitated proteins with anti- OTUD5, anti-p53 and anti-MDM2 antibodies. (D)Pull-down assay of His-tagged OTUD5 by GST fusion p53 protein bound to GSH-agarose beads, and subsequent detection by western with anti-His antibody. (E) Immunofluorescent detection of Flag-OTUD5 and Myc-p53. Nuclei are counterstained by DAPI.
Figure 2.
(A) The H1299 cells were cotransfected with 1 μg p53 along with increasing amounts of Flag-OTUD5. Cell lysates were prepared 24 h posttransfection and analyzed for the indicated proteins. Myc-GFP expression was used as an internal control for transfection efficiency. Actin expression shows equal loading of samples. (B) The U2OS cells were transfected with increasing amounts of Flag-OTUD5. Cell lysates were prepared 24 h posttransfection and analyzed for the indicated proteins. (C) Quantitative real time RT-PCR analysis of p53 mRNA level. RNA was extracted from U2OS-control and U2OS-OTUD5 cells. p53 mRNA was measured by quantitative RT-PCR, and levels relative to GAPDH mRNA are shown. Results are the means ± standard deviations of three independent experiments. (D) Half-life assay of endogenous p53 protein. U2OS cells were transfected with vector or Flag-OTUD5 then treated with 30 μg/ml cycloheximide for the indicated durations and protein levels were detected by western blotting using anti-p53, anti-Flag and anti-actin antibodies. (E) Quantification of the experiment. The values shown are obtained from three independent experiments and are normalized to the actin control. For each experimental condition, the signal at the start of the experiment was set to one. (F) Endogenous p53 levels in control and shOTUD5 U2OS cells were analyzed by western blotting with the anti-p53 antibody. Actin was used as loading control. (G) Quantitative real time RT-PCR analysis of p53 mRNA level. RNA was extracted from control and shOTUD5 U2OS cells. p53 mRNA was measured by quantitative RT-PCR, and levels relative to GAPDH mRNA are shown. Results are the means ± standard deviations of three independent experiments.
Figure 3.
(A) In vitro translated p53 was pre-incubated for 90 min with a bacterially expressed MDM2 and subsequently immunoprecipitated with an anti-p53 antibody. Immunoprecipitated p53 was then incubated with in vitro translated OTUD5, followed by western blotting with anti-UB to detect the ubiquitination of p53 and blotted with anti-p53, anti-OTUD5 and anti-MDM2 as loading control. (B) OTUD5 does not affect the auto-ubiquitination of MDM2 in vitro. Immobilized GST–Mdm2 protein was incubated with His-OTUD5, E1, E2 and Ub as indicated. (C,D) The H1299 cells were transfected with the indicated combinations of expression vectors. Cells, 24 h after transfection, were treated with MG132 (5 μM) for 3 h and then lysed. Cell lysates were immunoprecipitated with anti-HA antibody (against HA-p53) and immunoblotted with anti-Myc antibody (against Myc-ubiquitin).
Figure 4.
OTUD5 can help in the stabilization and activation of p53 in response to DNA damage.
(A) U2OS cells were infected with lentiviruses expressing either control or shRNA targeting OTUD5. After 48 h, cells were treated with 2.5μM Dox for indicated time. Cells were harvested and lysates were prepared for western blotting. The panels show immunoblots probed with the indicated antibodies. (B) U2OS-control and U2OS-shRNA cells were transfected with indicated combinations of expression vectors. Cells, 24 h after transfection, were treated with 2.5μM Dox for 3 h and MG132 (5 μM) for 3 h, then lysed. Cell lysates were immunoprecipitated with anti-HA antibody (against HA-p53) and immunoblotted with anti-Myc antibody (against Myc-ubiquitin). (C) Expression of Puma p21 Bax mRNA by quantitative RT-PCR. U2OS-control and U2OS-OTUD5-shRNA cells were treated with 2.5μM Dox for 12 h, and mRNA were extracted and subjected to quantitative RT-PCR.
Figure 5.
U2OS-control and U2OS-OTUD5-shRNA cells were treated with or without 2.5μM Dox as indicated. 24 h later, cells were harvested, stained with propidium iodide (PI), and the cell cycle distribution was determined by flow cytometry. The percentage of cells in various phases of the cell cycle is shown.