Mre11 nb100 142

The following antibodies were used:
For immunoprecipitation: MRE11 ab109623, 1 μl (Abcam); PRMT1 ab73246, 1 μl (Abcam); 53BP1 A300-272A, 1 μl (Bethyl); ATM ab32420, 3 μl (Abcam); GFI1 AF3540 2 μl (R&D Systems).
For immunoblotting: MRE11 ab109623, 1:10,000 (Abcam); PRMT1 ab12189, 1:2,000 (Abcam); 53BP1 A300-273A, 1:5000 (Bethyl); ATM ab32420, 1:5000 (Abcam); LaminB1 b-10 (mouse target only), 1:5000 (Santa-Cruz) or LaminB1 ab16048 (mouse and human target), 1:10,000 (Abcam); Asymmetric di-methyl arginine (ADMA) Asym-26 1:2000 (Epicypher); GFI1 AF3540 1:2000 (R&D Systems).
For immunofluorescence and FACS: γ-H2AX S139, 1:200 (Cell Signalling); ATM pS1981, 1:500 (Rockland 200-301-400); 53BP1 A300-272A, 1:500 (Bethyl); MRE11 nb100-142 1:500 (Novus Biologicals).

Cells were lysed in buffer, containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 1% Triton X-100, 20 mM glycerophosphate, 1 mM sodium orthovanadate, 5 mM EGTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. The samples were equalized according to the amount of protein, which was measured by the Bradford method, 500 µg of total protein was used. The lysates were incubated with Protein A agarose beads (1% bead slurry for one sample) at 4 °C for 1 h for the pre-clearing. The supernatant was transferred to the fresh tube and incubated with primary antibodies to Rad50 (1:300) (NB100-154, Novus Biologicals, Centennial, CO, USA) with gentle rocking at 4 °C overnight. The next day, Protein A agarose beads were added to the mixture (2% bead slurry for one sample) and incubated with gentle rocking at 4 °C for 1–3 h. After microcentrifuge for 5 min at 200× g, 4 °C, the pellet was washed three times with cell lysis buffer, resuspended with sample buffer for SDS-PAGE, heated to 95–100 °C for 5 min, and centrifuged for 1 min at 14,000× g. Then protein electrophoresis and immunoblotting were carried out, as described in the previous section. As primary antibodies, we used antibodies to Mre11 #NB 100-142 (Novus Biologicals, Centennial, CO, USA) (1:5000).