Anti 53bp1 nb100 304

The following antibodies were used for western blotting or immunoprecipitation: anti-XRCC4 (mouse, 611506, BD), anti-CtIP (sc5970, Santa Cruz Biotechnology), anti-XRCC3 (sc53471, Santa Cruz Biotechnology), anti-XLF (ab33499, Abcam), anti-tubulin (sc5286, Santa Cruz Biotechnology), anti-phospho-histone H3 (S10) (06-570, Millipore), anti-FLAG (1E6, Wako), anti-DNA ligase IV (this study) and anti-pS326 of XRCC4 (this study). Antibodies used for immunostaining were as follows: anti-53BP1 (NB100-304, Novus Biologicals), anti-Rad51 [61] (link), anti-XRCC4 (this study), and anti-DNA ligase IV (this study). For preparation of antibodies against XRCC4 and DNA ligase IV, full-length human XRCC4 tagged with hexahistidine and a 367-residue peptide containing the C-terminus of human DNA ligase IV tagged with hexahistidine were affinity purified from Escherichia coli with a nickel/cobalt column and used for immunization of rat or guinea pig, respectively. Anti-pS326 was raised in rabbits against a synthesized phosphopeptide, TLRNSpSPEDLFC. Post-immune IgG was affinity purified with this phosphopeptide and also titrated using a non-phosphorylated peptide, TLRNSSPEDLFC (custom-made by MBL Co., Ltd.). Immunization and preparation of antisera were carried out by MBL Co., Ltd.

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.