Anti atm ps1981

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.

Human mesenchymal stem cells were plated on μ-Slide 8 well ibitreat (Ibidi, Martinsried, Germany), fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 and blocked with 6% (wt/vol) BSA (Sigma-Aldrich) and 2.5% (vol/vol) normal goat serum (Sigma-Aldrich). Cells were then stained with the following primary antibodies: mouse monoclonal antibodies anti-phospho-Histone H2AX(Ser139) 1:500 (clone JBW301; Millipore, Billerica, MA, USA) and anti-ATMpS1981 1:300 (clone 10H11.E12, Rockland) or rabbit polyclonal antibody against 53BP1 1:500 (Novus Biological, Cambridge, UK) for 2 hrs at 4°C. Secondary antibodies were: Alexa 488 or Alexa 546 conjugated goat anti mouse and Alexa 546 conjugated goat anti-rabbit (all 1:500; Molecular Probes Inc.). Nuclei were stained with 0.1 μg/ml 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and slides mounted by using Mowiol solution (Calbiochem). Fluorescence images were obtained with a TCS-SPE, Leica Microsystem at 63× magnification. γH2AX foci quantification was performed counting at least 300 cells for each time-point.

For examination of phosphorylation of ATM after bleomycin treatment, lymyphoblastoid cells in logarithmic growth were treated in triplicate with 0, 10, and 30 μg/ml bleomycin for 1 h at 37°C. Immunoprecipitation was performed with the FISH-antibody (Anti-ATM, rabbit polyclonal, Novus) and magnetic beads (Dynal Biotech ASA/Invitrogen). The separated proteins were probed with anti-ATM pS1981 (Rockland, monoclonal, mouse), and reprobed with an anti-ATM antibody (Abcam Cambrige, UK) [74 ].