Anti rad51

Cells were grown on 35-mm coverslips and harvested at the indicated times after treatments. For immunofluorescence (IF,) after further washing with PBS, cells were pre-extracted with 0.5% Triton X-100 and fixed with 3% para-formaldehyde (PFA)/2% sucrose at room temperature (RT) for 10 min. For RAD51, IF cells were fixed in 4% PFA/PBS and subsequently with cold methanol for 20 min. Cells were permeabilized with 0.5% Triton X-100. After blocking in 3% bovine serum albumine (BSA) for 15 min, staining was performed with mouse polyclonal anti-RPA32 (Santa Cruz, 1:300), rabbit monoclonal anti-RAD51 (Bioss, 1:100), anti-SMARCAL1 (Abcam, 1:100) diluted in a 1% BSA/0.1% saponin in PBS solution, for 1 h at 37 °C in a humidifier chamber. Nocodazole-treated cells were blocked and fix with PTEMF (0.2% Triton X-100, 20 mM PIPES pH 6.8, 1 mM MgCl2, 10 mM EGTA and 4% formaldehyde) buffer. After blocking, coverslips were incubated for 1 h at RT with the indicated antibodies. After extensive washing with PBS, species-specific fluorophore-conjugated antibodies (Invitrogen) were applied for 1 h at RT followed by counterstaining with 0.5 mg/ml 4,6-diamidino-2-phenylindole (DAPI). Secondary antibodies were used at 1:200 dilution. A complete list of antibodies and dilutions is provided in Supplementary Table 2 and Souce data file.

Western blots were performed using standard methods. Blots were incubated with primary antibodies against: anti-MUS81 (Santa Cruz Biotechnology, 1:2000), Lamin B1 (Abcam, 1:10,000), anti-RAD52 (Santa Cruz Biotechnology, 1:500), anti-GAPDH (Millipore, 1:5000), anti-RAD51 (Bioss 1:1000), anti-SMARCAL1 (Bethyl 1:1000), anti-RPA32 (Santa Cruz Biotechnology 1:1000) and anti-ZRANB3 (Proteintech 1:1000). After incubations with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch, 1:30,000), the blots were developed using the chemiluminescence detection kit ECL-Plus (Amersham) according to the manufacturer’s instructions. Quantification was performed on blot acquired by ChemiDoc XRS+ (Bio-Rad) using Image Lab software, and values shown on the graphs represent a normalisation of the protein content evaluated through Lamin B1 or GAPDH immunoblotting. A complete list of antibodies and dilutions is provided in Supplementary Table 2 and as Souce data file.

Cells were lysed in RIPA buffer (Boston Bio Products, Ashland, MA, USA) supplemented with a cocktail of protease inhibitors (5892791001, Roche, Basel, Germany) and phosphatase inhibitor (4906845001, Roche). Protein quantification was determined using the Bradford protein determination assay (Bio-Rad). A 30 μg sample of protein was loaded and resolved on a 10% NuPAGE BIS-TRIS gel (Life Technologies), then transferred to a nitrocellulose membrane (Bio-Rad) before incubation with primary antibodies. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) antibody was obtained from Santa Cruz (sc-5282, Dallas, TX, USA) and anti-phosphorylated DNA-PKcs was purchased from Abcam (ab124918, Cambridge, MA, USA). Anti-RAD51 was purchased from BIOSS Antibodies (BSM-51402M, Woburn, MA, USA) and anti-β-actin was obtained from Cell Signaling Technologies (3700, Danvers, MA, USA). Gluc antibody was obtained from New England BioLabs (E8023). GAPDH antibody was obtained from Novus Biologicals (NB300–228, Centennial, Colorado, USA). Proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (#34077, Thermo Fisher Scientific).