IRTKS Regulates MDM2-Mediated p53 Ubiquitination Figure 6. IRTKS enhanced the interaction between p53 and MDM2. (A) Identification of p53, MDM2 and IRTKS within the same complex by co-immunoprecipitation assays with antibodies for p53 and MDM2. (B) Ectopic IRTKS enhanced p53-MDM2 interaction and the p53 ubiquitination in H1299 cells with low levels of MDM2. H1299 cells were co-transfected with the plasmids for expressing p53, varying amounts of MDM2 and IRTKS or control plasmid. Immunoprecipitations with anti-p53 antibody (DO-1) were analyzed by Western blotting with p53 antibody (FL393) and MDM2. The long time exposal of the Western blotting with anti-p53 antibody indicated p53 ubiquitination (the third panel). (C) Knockdown of IRTKS reduced the p53-MDM2 interaction and p53 ubiquitination in H1299 cells at low levels of MDM2. After pre-transfected with IRTKS siRNA or control siRNA for 24 h, H1299 cells were transfected with the plasmids for expressing p53 and varying amounts of MDM2. The p53-MDM2 interactions and p53 ubiquitination were analyzed. doi:10.1371/journal.pone.0023571.g006 Cell culture Human HT1080, U2OS, SAOS-2, H1299 and HEK 293 cells (ATCC) were cultured at 37uC in DMEM supplemented with 10% fetal bovine serum (FBS). p53–Mdm2 double knockout mouse embryonic fibroblast cell line was kindly provided by Prof. Long Yu (Fudan University). Cells were transfected using FuGENE HD or FuGENE 6 by the manufacturer’s protocol (Roche). Short interfering RNA (siRNA) oligonucleotides were transfected into cells using Lipofectamine 2000 by the manufac- turer’s instructions (Invitrogen). The IRTKS siRNA (siIRTKS) sequence was 59-GCUUAAGCAAAUCAUGCUU-39. The same sequence with two nucleotides changed (59-GCUUGAG- CAAAUCAUACUU-39) was used as a specific RNAi control(- siIRTKSm). The p53 siRNA sequence was 59-GACUCCAGUG- GUAAUCUAC-39 and MDM2 siRNA sequence was 59- CCACCUCACAGAUUCCAGC-39. Luciferase siRNA (59- CUUACGCUGAGUACUUCG ATT-39) was used as a control siRNA(siLUC). Apoptosis assay HT1080 cells transfected with FLAG-IRTKS or IRTKS siRNA were UV-irradiated (60 J/M2). The cell apoptosis was detected by using Annexin V-FITC apoptosis detection kit (Pharmingen) according to the manufacturer’s instruction. Cells that stain positive for Annexin V-FITC and negative for propidium iodide were counted as apoptotic cells. Immunofluorescence microscopy Cells cultured on coverslips were washed twice with cold PBS. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 5% bovine serum albumin (BSA) and incubated with anti-IRTKS and anti-p53 or anti-MDM2 antibodies as indicated, followed by incubation with Alexa-546 conjugated anti-rabbit IgG and Alexa- 488 conjugated anti-mouse IgG antibody (Invitrogen). The cells were mounted with DAPI-containing medium (Sigma) and the images were acquired with a confocal microscope. Immunoprecipitation and Western blotting Whole cell lysates were prepared in the lysis buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100, 16 complete protease cocktail, 5 mM sodium vanadate, and 10% glycerol). The lysates were then immunoprecipitated with the indicated antibodies and isotype-matched control antibodies plus protein G Sepharose (Roche) for at least 4 h or overnight. Beads were washed three times with the lysis buffer, and boiled in 26 loading buffer. Protein samples were resolved by SDS–PAGE and transferred onto nitrocellulose membrane, which was blocked in 5% skim milk in PBS-Tween and probed with the indicated antibodies. The membrane was scanned by Odyssey infrared imaging system (LI-COR) at a 700–800 channel wavelength. Subcellular fractionation Nuclear and cytoplasmic fractions were prepared using NE- PERH Nuclear and Cytoplasmic Extraction Reagents (Pierce biotechnology) according to the manufacturers’ protocol. Purity of fractions was determined by western blotting with anti-HDAC1 and anti-Tublin (Santa Cruz Biotechnology) antibodies. In vivo ubiquitination and neddylation assay Cells were transfected with His-ubiquitin, HA-ubiquitin or HA-NEDD8 and other indicated plasmids. Twenty hours after transfection, cells were treated with 25 mM MG132 for 4–6 h and scraped into PBS and pelleted by centrifugation. For transfections with His-ubiquitination, cells were resuspended in 1 ml of PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23571