Anti mouse plus and anti rabbit minus pla probes

EdU-PLA to detect proteins at nascent DNA was performed as described (Taglialatela et al., 2017 (link)), with minor modifications. Briefly, cells were pulsed with 10 μM EdU for 10 min cells before being permeabilised with nuclear extraction buffer (10 mM PIPES, 20 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100). Where appropriate, cells were exposed to 4 mM HU for 5 hr before pre-extraction. Cells were then fixed with 3.6% paraformaldehyde for 10 min at room temperature and blocked with ADB (Antibody Dilution Buffer; 3% BSA in PBS) overnight at 4°C. EdU was conjugated to biotin by incubating cells in Click reaction buffer for 1 hr at room temperature containing 10 μM Diazo-biotin Azide, 10 mM sodium ascorbate, and 1 mM copper (II) sulfate in PBS. Following the Click reaction, cells were blocked in ABD before incubated in primary antibodies before proceeding with proximity ligation using a Duolink Detection Kit in combination with anti-Mouse PLUS and anti-Rabbit MINUS PLA Probes (Sigma Aldrich) according to the manufacturer’s instructions. Cells were imaged as below and the number of PLA signals per nuclei quantified.

HeLa cells were either mock treated or treated with 5 Gy of IR, and were fixed at 2 h in 4% formaldehyde for 15 min and 100% formaldehyde for 5 min, followed by permeabilization with 0.5% Triton X-100 for 15 min at room temperature. In situ proximity ligation assay (PLA) was performed using Duolink PLA technology (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, coverslip was blocked in Duolink blocking buffer for 1 h at room temperature and then incubated with the two primary antibodies. The primary antibodies used were as follows: rabbit anti-RAD51 (ab63801, Abcam, 1:100) and mouse monoclonal anti-TOPORS (H00010210-M01, Abnova, 1:100). The negative control used only one primary antibody. The coverslips were washed twice in PBS for 5 min; anti-mouse PLUS and anti-rabbit MINUS PLA probes (DU920004 and DUO92002, respectively, Sigma-Aldrich) were coupled to the primary antibodies for 1 h at 37°C. Next, amplification was performed using the ‘Duolink In Situ Detection Reagents Red’ (DUO92001, DUO92005, Sigma-Aldrich). Finally, coverslips were mounted using Vectashield mounting media (Vector Laboratories) containing DAPI and imaged on a Carl Zeiss LSM 900 confocal microscope.