Anti rad50

1–2 mg of protein from cytosolic and nuclear fractions or NP40 extracts (420 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% NP40, 10% glycerol, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin and 1 μg/ml leupeptin, centrifuged and then diluted to 150 mM NaCl) were incubated with normal mouse (sc-2025) or rabbit (sc-2027) sera (Santa Cruz Biotechnology) for 30 min and subsequently with protein G or A-sepharose beads (GE Healthcare), respectively, for 1 h at 4 °C. After centrifugation, the beads were discarded and the supernatants incubated for 2 h with anti-FBXW7 (A301-720A, Bethyl laboratories, Montgomery, TX, USA), anti-Flag (F3165, Sigma-Aldrich), anti-MRE11 (#4847, Cell Signaling Technology), anti-NBS1 (#14956, Cell Signaling Technology), anti-RAD50 (NB110-60483, Novus Biologicals) or with normal mouse or rabbit sera, followed by protein G or A-sepharose beads for 1 h. The beads were washed, and the bound proteins were solubilized by adding SDS sample buffer and heated at 95 °C for 5 min.

Cells were lysed in RIPA Lysis buffer (Millipore) supplemented with 0.1% SDS, protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktails (Sigma). Samples were prepared using NuPage LDS Sample buffer (Invitrogen) with 100 mM DTT, and proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using the NuPage MOPS system (Invitrogen). Antibodies used were as follows: anti-Mre11 (Novus Biological, NB 100–142, 1:2000), anti-NBS1 (p95/NBS1, Cell Signaling #3002, 1:1000), anti-Rad50 (Novus Biological, NB 100–154, 1:500), anti-pATM S1981(Cell Signaling #4526, 1:500), anti-pATR S1989 (Kerafast, 1:500), anti-Chk1 S317 (Cell Signaling, #2344, 1:400), anti-Chk1 S345 (Cell Signaling, #2348, 1:400).