Mouse anti mre11

Cells were cultured onto 22 × 22 coverslip or eight-well Nunc chamber slides. To detect MRE11, WRN, BLM, RPA32 or pS4/8-RPA32 foci, we performed pre-extraction for 5 min on ice in CSK buffer followed by fixation with 4% PFA/PBS for 10 min, permeabilization in 0.5% Triton X100/PBS for 15 min and blocking in 10% FBS/PBS for 1 h, as described elsewhere22 (link). Cells were incubated with specific primary antibody: rabbit anti-WRN (Abcam); rabbit anti-phosphoS4/8RPA32 (Bethyl); mouse anti-RPA32 (Calbiochem); mouse anti-MRE11 (Abcam); and goat anti-BLM (Santa Cruz), for 2 h at room temperature diluted in 1%BSA/PBS, followed by species-specific fluorescein-conjugated secondary antibodies (Alexa Fluor 594 Anti-Rabbit or Alexa Fluor 488 Anti-Mouse), and counterstained with 0.5 μg ml⁻¹ 4,6-diamidino-2-phenylindole (DAPI). Slides were analysed with Eclipse 80i Nikon Fluorescence Microscope, equipped with a VideoConfocal (ViCo) system. For each time point, at least 100 nuclei were scored at × 40. Detailed information on antibodies and their usage can be found in Supplementary Information Online.

The in situ PLA in combination with immunofluorescence microscopy was performed using the Duolink II Detection Kit with anti-Mouse PLUS and anti-Rabbit MINUS PLA Probes, according to the manufacturer's instructions (Sigma-Aldrich).
To detect proteins, we used rabbit anti-WRN (Abcam) and mouse anti-MRE11 (Abcam) antibodies. Detailed information on antibodies and their usage can be found in Supplementary Information Online.