Figure S2 IRTKS interacted with p53. (A) HT1080 cells were pretreated with the proteasome inhibitor MG132. The endogenous interaction of IRTKS and p53 was analysed by immunoprecipitation assay with anti-p53 antibody. (B) The fine- mapping of the interaction sites of p53 binding to IRTKS by GST-pulldown assays. (TIF) Figure S3 IRTKS could not affect the phosphorylation and acetylation of certain amino acid residues of p53. (A) The effect of IRTKS overexpression on p53 phosphorylation. HT1080 cells transfected with IRTKS or control plasmid were untreated, UV-irradiated (60 J/M2) or treated with doxorubicin (DOX, 1 mM) for 20 h and collected in lysis buffer. The cell lysates were immunoprecipitated (IP) with anti-p53 antibodies and analyzed by Western blotting using anti-phospho-p53 (S15, S37, S46) and anti-p53 antibodies. (B) The effect of IRTKS depletion on p53 phosphorylation. HT1080 cells transfected with IRTKS siRNA were treated with UV irradiation and harvested at indicated time. p53 phosphorylation were analyzed by Western blotting using anti-phospho-p53 (S15, S20 and S392). (C) and (D) HT1080 cells with overexpression (C) or knockdown (D) of IRTKS were UV-irradiated for 20 hr. The cells treated with trichostatin A (10 nM) for 4 hr and collected in lysis buffer. The cell lysates were immunoprecipitated (IP) with anti-p53 antibodies and analyzed by Western blotting using anti-acetyl-p53 (K382) and anti-p53 antibodies. (TIF) Figure S4 IRTKS enhanced p53 ubiquitination. (A) IRTKS enhanced ubiquitination of endogenous p53. HT1080 cells transfected with IRTKS or control plasmid were untreated, UV-irradiated (60 J/M2) or treated with doxorubicin (DOX, 1 mM) for 20 h. The cells were boiled in denaturing lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% SDS). The denaturing lysates were diluted with dilution buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) until the concentration of SDS reached 0.2% and immunoprecipitated (IP) with anti-p53 antibodies. The immunoprecipitates were analyzed by Western blotting using anti-ubiquitin and anti-p53 antibodies. (B) Knock- down of IRTKS inhibited p53 ubiquitination. HT1080 cells transfected with IRTKS siRNA were treated with UV or DOX. References 1. Kruse JP, Gu W (2009) Modes of p53 regulation. Cell 137: 609–622. 2. Bode AM, Dong Z (2004) Post-translational modification of p53 in tumorigen- esis. Nat Rev Cancer 4: 793–805. 3. Kruse JP, Gu W (2008) SnapShot: p53 posttranslational modifications. Cell 133: 930–930 e931. 4. Brooks CL, Gu W (2006) p53 ubiquitination: Mdm2 and beyond. Mol Cell 21: 307–315. 5. Sigismund S, Polo S, Di Fiore PP (2004) Signaling through monoubiquitination. Curr Top Microbiol Immunol 286: 149–185. 6. 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