Phosphorylated s1981 atm

Cells were 1) fixed in 4% formaldehyde (MilliporeSigma) for 15 min, 2) permeabilized by 0.25% Triton-X (MilliporeSigma) for 10 min, and 3) blocked with 5% bovine serum albumin (BSA) (MilliporeSigma) for 30 min, all at room temperature. Then cells were incubated in primary antibodies overnight at 4^o. The antibodies used include lamin-A/C (Santa Cruz and Cell Signaling), lamin-B (Santa Cruz), KU80 (Cell Signaling), γH2AX (MilliporeSigma), 53BP1 (Abcam), phosphorylated S1981 ATM (Abcam), topoisomerase IIα (Santa Cruz), BRCA1 (Santa Cruz), BRCA2 (MilliporeSigma), and RPA1 (Santa Cruz). Finally, cells were incubated in secondary antibodies (Thermo Fisher) for 1.5 h, and their nuclei were stained with 8 µM Hoechst 33342 (Thermo Fisher) for 15 min, all at room temperature. Cells were mounted with Prolong Gold antifade reagent (Invitrogen, Life Technologies). Epifluorescence imaging was performed on an Olympus IX71—with a 40×/0.6 NA objective—and a digital electron-multiplying charge-coupled device (EMCCD) camera (Cascade, Photometrics). For confocal imaging, we used a Leica TCS SP8 system, equipped with either a 63×/1.4 NA oil-immersion or a 40×/1.2 NA water-immersion objective. Superresolution images were taken using a Leica TCS SP8 STED 3X system with a 100×/1.4 NA oil-immersion objective. ImageJ (Schneider et al., 2012 (link)) was used to quantify the resulting images.