Immunoprecipitation of 53BP1 Protein

Immunoprecipitation was performed using Dynabeads Protein G (Thermo Fisher Scientific, cat#: 10004D) according to the following procedure^{33 (link)}. Briefly, nuclear fractions were obtained by NE-PER nuclear extraction kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Nuclear samples were pre-cleared by incubation with Dynabeads Protein G at 4 °C for 1 h in IP buffer (20 mM Tris pH7.5, 150 mM NaCl, 1% Triton-X 100, and protease inhibitor (Nacalai tesque)) and then incubated with 3 μg of antibodies (53BP1, cat#: NB100-304 Novus biologicals) at 4 °C for overnight. The samples were incubated with 30 μl of Dynabeads Protein G at room temperature for 2 h. Immunoprecipitated proteins were analyzed by immunoblotting as already described.