Mab3802

The foci assay for γH2AX, 53BP1, and Trf1 was performed as previously described [26 (link)]. Briefly, cells grown on coverslips were fixed, blocked, and stained for γH2AX and 53BP1 or γH2AX and Trf1. Foci for γH2AX and 53BP1 were assessed by LSM using a single focus plane; for γH2AX and Trf1 quantification, the whole cell was screened, and images were stacked. For each experiment, at least 100 cells were analyzed. Primary antibodies used were γH2AX (1:500, rabbit, Cell Signaling Technology, MA, USA, mAb #9718S), γH2AX (1:500, mouse, Merck Millipore, Darmstadt, Germany, 05-636), 53BP1 (1:500, mouse, Sigma Aldrich, Karlsruhe, Germany, MAB 3802), and Trf1 (1:500, mouse, Abcam, Cambridge, UK, ab10579). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse (1:1000, Thermo Fisher Scientific Invitrogen, Waltham, USA, A11017) and Cy3 goat anti-rabbit (1:1000, Abcam, ab97075). The software ImageJ (Wayne Rasband, NIH, Bethesda, MD, USA) was used to count foci and measure colocalization.

Primary antibodies: γH2A.X (Millipore, Merck 05–636, 1:1000), γH2A.X (Cell Signalling Technology, #9718, 1:1000), human CREST serum (AntibodiesInc, SKU 15–234, 1:800), R-loops (S9.6, Absolute Antibody, Ab01137-23.0, 1:400), TRF2 (Abcam, ab13579, 1:500), BRCA1 (Santa-Cruz Biotech, sc-6954 (D-9), 1:250), 53BP1 (Merck, MAB3802, 1:1000), 53BP1 (Abcam,ab36823, 1:1500), DAXX (Cell Signaling Technology, #4533, 1:25), a-tubulin (Proteintech, 66031-1-Ig, 1:750), ATRX (Abcam, ab97508, 1:500). Secondary antibodies: anti-mouse AlexaFluor 488 (Invitrogen, A11001, 1:500), anti-mouse AlexaFluor 555 (Invitrogen, A21422, 1:500), anti-mouse AlexaFluor 647 (Invitrogen, A-21235, 1:500), anti-rabbit AlexaFluor 488 (Invitrogen, A-11008, 1:500), anti-rabbit AlexaFluor 555 (Invitrogen, A21428, 1:500), anti-rabbit AlexaFluor 647 (Invitrogen, A21244, 1:500), anti-human AlexaFluor 647 (Invitrogen, A-21445, 1:500).