Immunoblotting, immunoprecipitation, and Strep-Tactin pull-downs were performed as previously described (Mailand et al., 2006 (link), 2007 (link)). In brief, cells were lysed in EBC buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% NP-40) or denaturing buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 0.5% sodium deoxycholate, and 0.5% SDS) supplemented with protease and phosphatase inhibitors and incubated on ice for 10 min, and lysates were cleared by centrifugation for 10 min at 20,000 rpm. Lysates were incubated with FLAG agarose (Sigma-Aldrich) or Strep-Tactin Sepharose (IBA BioTAGnology) for 1.5 h on an end-over-end rotator at 4°C, washed five times with EBC buffer or denaturing buffer, and resuspended in 2× Laemmli sample buffer. To analyze binding of RNF169 to ubiquitin chains, cells transfected with S-FLAG-Strep–tagged RNF169 constructs were lysed in denaturing buffer containing protease inhibitors and subjected to Strep-Tactin pull-down. Bound complexes were washed three times in denaturing buffer followed by two washes in EBC buffer and incubated with K48- or K63-linked polyubiquitin chains (Boston Biochem) for 2 h at 4°C. After thorough washing, immobilized material was resolved by SDS-PAGE and subjected to immunoblotting. For subcellular fractionation, the Subcellular Protein Fractionation kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Rabbit polyclonal antibody to RNF169 (Eurogentec) was raised against the peptide RRSQPERCRPRRDGGA, corresponding to amino acids 134–149 in human RNF169, and affinity purified. Other antibodies used in this study included rabbit polyclonals to 53BP1, BRCA1, SP1, Cyclin A, HA (Santa Cruz Biotechnology, Inc.), Myc (Abcam), NF-κB–p65, histone H2A, and γ-H2AX (Cell Signaling Technology), mouse monoclonals to FLAG (Sigma-Aldrich), His6 (Takara Bio Inc.), ubiquitin, and GFP (Santa Cruz Biotechnology, Inc.), conjugated ubiquitin (FK2; Enzo Life Sciences), Cyclin B (BD), and Plk1 (Invitrogen), and goat polyclonal to MCM6 (Santa Cruz Biotechnology, Inc.). Rabbit polyclonal antibodies to RNF168 and RAP80 were gifts from D. Durocher (Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada) and X. Yu (University of Michigan, Ann Arbor, MI), respectively. The sheep polyclonal MDC1 antibody was a gift from S. Jackson (Gurdon Institute, University of Cambridge, Cambridge, England, UK).