P53 s15p

Samples were loaded on 4–12% NuPAGE Bis-Tris Gel (Novex Life Technologies) and transferred to a polyvinylidene difluoride membrane (0.45 μm, Immobilon). Membranes were blocked with 5% BSA in PBS for 1 h at room temperature and incubated with primary antibodies diluted 1:1,000 in blocking buffer overnight at 4 °C. Primary antibodies were: H3K9me1 (Upstate, 07-450), H3K9me3 (Millipore, 07-442) mouse anti-H3 (Abcam, ab10799), γH2A.X (Millipore, 05-636), Chk1p (Cell Signaling, 2344), Chk1 (DCS-310 (ref. ^{98 (link)}), p53-S15p (Cell Signaling, 12571), p53 (Sigma-Aldrich, mouse monoclonal antibody, clone DO-1) and β-actin (Sigma-Aldrich, A5316). Membranes were washed in 0.1% Tween-20 in PBS on the following day, followed by incubation with secondary antibody coupled to near-infrared dyes CF 680/CF 770 (1:10,000). Antibodies were visualized using an Odyssey CLx infrared scanner (LiCor).

Primary antibodies used were: p53 (CM5, Leica) 1:1000 dilution, p53-s15p (#9284, Cell signaling) 1:1000 dilution, FL393 (Santa Cruz) 1:400 dilution, pH2AX (ab11174, abcam, 1:400 dilution). Secondary goat anti-mouse or anti-rabbit antibodies conjugated to AF555 or AF647 were purchased from Invitrogen and used at 1:1000 dilution.

For Western blot, proteins were extracted from 10 mg of crushed tumor in buffer (6 M urea, 5 mM NaF, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 0.5% TritonX100, 1 mM activated sodium orthovanadate, 1X protease inhibitors). Proteins (30 μg) were separated in 8% or 12.5% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, St Quentin-en-Yvelines, France). We used the following antibodies: p53-S15P (9284, Cell Signalling, Ozyme, St-Quentin-en-Yvelines, France), γH2AX (2577, Cell Signalling), p21 (6246, Santa Cruz, Clinisciences, Nanterre, France), and GAPDH (25778, Santa Cruz). VEGF level was determined by ELISA (Raybiotech, Tebu-bio SA, Le Perray-en-Yvelines, France) according to the manufacturer's instructions.