Anti p53 pser15

Samples were treated with lysis buffer [Tris–HCl 50mM pH7.4, 10% NP-40, 0.25% NaDesoxycholate, EDTA 1mM, NaCl 150mM, PMSF 1mM, protease inhibitor cocktail (Roche)] and loaded onto pre-cast 4–12% gradient acrylamide gels (NuPAGE, Invitrogen). After electro-blotting, filters were incubated with anti-AKTIP (Sigma), anti-TRF2 (Novus Biologicals), anti-actin-HRP conjugated (Santa Cruz), anti-cyclin A (Santa Cruz), anti-cyclin B (Santa Cruz), anti-cyclin E (Upstate Biotechnology), anti-p53-pSer15 (Cell Signaling Technology), anti-p53 (DakoCytomation), anti-ATM-pS1981 (Rockland Immunochemicals), anti-ATM (Genetex), anti-ChK1-PSer345 (Cell Signaling Technology), anti-AKT (Cell Signaling Technology), anti-PCNA (Santa Cruz), anti-RPA70 (Santa Cruz), or anti-TRF1 (Santa Cruz). Filters were then incubated with appropriate HRP-conjugated secondary antibodies (Santa Cruz), which were detected using the enhanced chemiluminescence system (ECL plus, Amersham). Signals were quantified with Image J software.

Cells or tumors were lysed with RIPA buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Samples were separated on a 10% acrylamide gel and transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in TBS containing 5% non-fat milk and 0.05% Tween 20 solution and incubated with primary antibody (Ab). The following antibodies were used for western blot: anti-Zta (Argene, Verniolle, France), anti-p53 (Novocastra, Buffalo Grove, IL, USA), anti-p53 pSer15 (Cell Signaling Technology, Beverly, MA, USA), anti-ATM (Cell Signaling Technology), anti-ATM pSer1981 (Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich)

Equal quantities of proteins (10 to 30 µg) were run on Mini-PROTEAN TGX Stain Free precast polyacrylamide 4–20% gels (BIORAD) in Tris glycine-SDS buffer (BIORAD). Proteins were transferred on Immobilon-P PVDF membranes (Millipore) in Tris glycine-Ethanol 20% buffer (BIORAD). Stain Free technology allows visualizing total proteins without use of any dye. The following antibodies were used at dilutions from 1/200 to 1/1000: anti-caspase 9 (32539, Abcam, Cambridge, UK), anti-cleaved caspase 3 (5A1E, Cell Signaling, Danvers, MA, USA), anti-PARP (Cell signaling 92845), anti-FGF1 (R&D AB-32-NA, Minneapolis, MN, USA) and anti-FGF1 (Cat.No.010-24161, WAKO, Osaka, Japan), anti-p53 DO1 (Santa Cruz Sc-126), anti-p53pSer15 (Cell signaling 92845), anti-PUMA N-19 (Santa Cruz Sc-19187), anti-Bax I-19 (Santa Cruz Sc-930), anti-p21 C-19 (Santa Cruz Sc-397-G) and anti-TOM40 H-300 (Santa Cruz Sc-11414). Secondary antibodies were HRP coupled (Jackson Immunoresearch, West Grove, PA, USA) and the revelation was performed using Clarity Western ECL Blotting Substrate (BIORAD). Secondary antibodies were HRP coupled (Jackson Immunoresearch) and the detection was performed using Clarity Western ECL Blotting Substrate (BIORAD). The chemiluminescent signal was captured by Chemidoc (BIORAD) and quantification was performed with the ImageLab software (BIORAD).