Immunofluorescence assay

Immunoflorescence of 53BP1 was carried out as previously reported.³⁵ In brief, cells were fixed in 4% paraformaldehyde for 10 min and in methanol at ¯20˚C for 20 min, permeabilized in PBS with 0.5% Triton X¯100 for 10 min, blocked with 5% skim milk for 2 h and stained with the primary antibody rabbit anti¯53BP1 antibody (Abcam, ab36823, UK) for 2 h at room temperature. The bound antibody was visualized using Alexa Fluor® 594 anti¯rabbit antibody (Molecular Probes, A-11037, USA), and cell nuclei were counterstained with DAPI solution (Invitrogen, {"type":"entrez-protein","attrs":{"text":"P36941","term_id":"549090","term_text":"P36941"}}P36941). Images were captured and 53BP1 foci were counted using a fluorescence microscope (Leica, Wetzlar, Hesse-Darmstadt, Germany), and a minimum of 100 cells were scored for each sample.