Mre11 ab214

For the immunofluorescence staining of pATM cells were fixed at room temperature in 2% formaldehyde (15 minutes) and permeabilized in 0.5% triton X-100 (10 minutes). MRE11 staining is applied after extraction of soluble proteins as described previously [21] (link). For staining of poly-ADP-ribose (PAR) cells were fixed following a standardized protocol [27] . Staining of PAR (clone H10, Oncogene, San Diego, USA) and pATM (anti-phospho-(Ser1981)-ATM, Rockland, Gilbertsville, USA) was performed with a dilution of 1∶200 and 1∶750 for MRE11 ab214 (Abcam, Cambridge, UK) in 0.4% BSA in PBS for one hour at room temperature. Primary antibodies were detected with 5 μg/ml Alexa 488 or 568 goat anti-mouse IgG conjugate (Molecular Probes, Leiden, The Netherlands) (1 hour, room temperature) respectively and counterstained with 1 μg/ml DAPI (20 minutes, room temperature).

Knockdowns were performed using the INTERFERin transfection kit (PeqLab, Erlangen, Germany) according to the standard protocol. SiRNA sequences can be found in tab. S1. Efficiency of each knockdown was analyzed by western blot 48 h after treatment following a standardized protocol [26] (link) and quantified using ImageJ software. When applicable further knockdown verification was done by immunofluorescence assays. Antibodies used were: ACF1/BAZ1A (Bethyl Laboratories, Montgomery, USA), MRE11 ab214 (Abcam, Cambridge, UK), NIPBL (Santa Cruz, Heidelberg, Germany), PARG (Merck Millipore, Billerica, USA), PARP (Cell Signaling, Danvers, USA), SMC1 (Cell Signaling, Danver, USA). 10 mM caffeine (Sigma Aldrich, Hamburg, Germany) was used for unspecific inhibition of ATM and 15 μM KU55933 (Merck Millipore, Billerica, USA) for specific ATM kinase inhibition. PARP1 was inhibited by 10 μM PJ34 (Sigma Aldrich, Hamburg, Germany). Inhibition of all proteins was started two hours before irradiation and efficiency of inhibition was analyzed by immunofluorescence staining as described in the results. Depletion of ATP was carried out by 10 mM sodium azide and 50 mM 2-desoxyglucose diluted in culture medium 30 minutes prior to irradiation, resulting in partial depletion to ensure the formation of repair foci.