IRTKS Regulates MDM2-Mediated p53 Ubiquitination Figure 7. MDM2 induced polyubiquitination and degradation of IRTKS. (A) Downregulation of IRTKS by DNA damage in HT1080 cells. Cells were treated with UV-irradiation (UV, 60 J/M2) or etoposide (ETO, 30 mM), collected at indicated time and analysed by Western blotting. (B) Ectopic MDM2 decreased endogenous IRTKS expression. The expression of endogenous IRTKS in HT1080 cells with MDM2 overexpression were analysed by Western blotting. (C) MDM2 directly mediated IRTKS ubiquitination. In vitro ubiquitination assays were performed with recombinant His-IRTKS and varying amounts of His-MDM2, and analyzed with anti-IRTKS antibody by Western blotting. (D) MDM2 induced IRTKS ubiquitination in vivo. In vivo ubiquitination assay was performed with co-transfection of HA-Ubiquitin, FLAG-IRTKS and varying amounts of MDM2 in H1299 cells. The cells were treated with 25 mM MG132 for 4 h before lysis. FLAG-IRTKS was immunoprecipitated with FLAG-beads and ubiquitination of IRTKS was analyzed by western blotting with HA antibody. (E) DNA damage dissociated IRTKS from p53 and enhanced the IRTKS-MDM2 interaction. U2OS cells were treated with 25 mM MG-132 for 4 h and subsequently with 30 mM etoposide as indicated. The p53-IRTKS and MDM2-IRTKS interactions were analyzed. doi:10.1371/journal.pone.0023571.g007 denaturing buffer (6 M guanidinium-HCl, 0.5% Triton, 10 mM imidazole, 20 mM Tris-HCl [pH 8], 0.5 mM DTT, and 0.5 mM iodoacetamide) with mild sonication. His–Ub conjugated proteins were purified by nickel chromatography (Ni-NTA Agarose; Qiagen). Nickel bound ubiquitinated proteins were subjected to Western blotting analysis using anti-p53 Ab (DO-1). For transfections with HA-ubiquitin or HA-NEDD8, cells were resuspended in 1% SDS in TBS and boiled twice for 5 min each time. The lysated were diluted to a solution with final concentrations of 0.2% SDS and 1% Triton X-100, and immunoprecipitated with anti-HA antibody (Sigma). The ubiqui- tinated or neddylated proteins were analyzed with Western blotting by using anti-p53 Ab (DO-1). In vitro ubiquitination assay Purified recombinant proteins as indicated were incubated in 20 ml of reaction buffer (50 mM Tris-HCl pH 7.6, 1 mM DTT, 2 mM ATP and 5 mM MgCl2), in the presence of 50 ng of E1 and 100 ng E2 (UbcH5a) enzymes and 10 mg of ubiquitin (Boston Biochem, Boston, MS). After incubation at 32uC for 1 h, the reaction products were analyzed by SDS-PAGE followed by Western blotting. Supporting Information Figure S1 IRTKS inhibited apoptosis. (A) Overexpression of IRTKS inhibited UV-induced apoptosis. HT1080 cells were transfected with pCMV-FLAG-IRTKS for overexpression of IRTKS, UV-irradiated (60 J/M2) and analysed by FACS with Annexin V FITC/PI staining. For evaluation of cells undergoing apoptosis, cells were stained by both Annexin V-FITC and PI, and the PI-positive cells were excluded from the measurement. (B) Knockdown of IRTKS enhanced UV-induced apoptosis in HT1080 cells. HT1080 cells were transfected with IRTKS siRNA and control siRNA, UV-irradiated and analysed by FACS with Annexin V FITC/PI staining. (C) HT1080 cells were transfected with IRTKS siRNA and control siRNA, UV-irradiated and analysed with anti-cleaved PARP, anti-p53 and anti-IRTKS by Western blotting at indicated time points. Note that the cleaved PARP was increased earlier in the cells with IRTKS knockdown and IRTKS was decreased after UV irradiation. (D) Overexpres- sion of IRTKS reduced the apoptosis induced by p53. SAOS-2 cells were examined by phase contrast microscopy after infection with Ad-p53, Ad-IRTKS and control virus: AdGFP, for 48 h. (E) Knockdown of IRTKS increased the apoptosis induced by p53. (TIF) PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23571