Anti trf2 clone 4a794

Cells grown on poly-L-lysine-coated coverslips (Becton Dickinson) were placed in cytobuffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM pipes pH 6.8) for 30 s, washed in cytobuffer with 0.5% Triton X-100 for 30 s, washed in cytobuffer for 30 s for and then fixed for 10 min in 4% paraformaldehyde in PBS. Cells were blocked with 100% FBS for 1 h at room temperature. Cells were incubated with primary antibody dissolved in Dako antibody Diluent (Dako) overnight in a humid chamber at 4 °C. Next day, coverslips were washed 3 times for 30 min in 1 × PBS containing 0.1% Tween-20. Cells were incubated with Alexa secondary antibody (Life Technologies, A11017) dissolved in Dako antibody Diluent (Dako) for 1 h in a humid chamber at room temperature. Cells were washed three times for 30 min in 1 × PBS. Samples were mounted in Prolong with Dapi (Invitrogen). Signals were visualized in a confocal ultraespectral microscope SP5-WLL (Leica). The following antibodies were used: anti-phospho-Histone γH2AX (05-636, Millipore) diluted 1:200, anti-53BP1 (NB-100-304, Novus) diluted 1:200, anti-TRF1 (TRF78, Abcam) diluted 1:200 and anti-TRF2 (clone 4A794, Millipore) diluted 1:200.

For Western blot analysis, cells were collected and lysed in a proper buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 250 mM NaCl, 0.1% Triton) completed with inhibitors of protease (ThermoScientific, A32953) and phosphatase (ThermoScientific, 88667). Total proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL, USA). Membranes were probed with the following primary antibodies: mouse mAb anti-TRF2 (clone 4A794, Millipore, Billerica, MA, USA) and mouse mAb anti-β-actin (clone AC-15, Sigma-Aldrich, St. Louis, MO, USA).