Complete edta free proteinase inhibitor

Co-immunoprecipitation analyses were performed as for I-DIRT pull-down with the only exception that protein elution from magnetic beads was performed by incubation with NuPage LDS Sample buffer (Thermo Fisher) supplemented with 50mM DTT for 10 min at 70C. Where indicated, 10 μM ATMi KU55933 (Tocris Bioscience) was added 1h before irradiation. Western blot analysis of protein levels was performed on whole cell lysates prepared by lysis in RIPA buffer supplemented with Complete EDTA free proteinase inhibitor (Roche). The antibodies used for WB analysis are: anti-Rif1 (Di Virgilio et al., 2013 (link)), anti-Flag M2 (Sigma-Aldrich), anti-ZMYND8 (Sigma-Aldrich), anti-γH2AX (Millipore), anti-β Actin (Sigma-Aldrich), and anti-53BP1 (Bethyl).

Total cell extracts were obtained by lysing cells in 150 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% octylphenoxypolyethoxyethanol (IGEPAL), 1% Triton, 1 mM EDTA, 0.5% deoxycholate with cOmplete EDTA-free proteinase inhibitors (Roche, Indianapolis, IN), and Benzoase DNase (Novagen). For immunoprecipitations, cells were lysed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, 5 mM EDTA, and 0.5% IGEPAL with Benzoase DNase and protease inhibitors. Successive washes were carried out with reducing IGEPAL content to a final concentration of 0.01%, with final washes using only Tris-HCl, NaCl, EDTA, and 10% glycerol. Primary antibody was generally incubated overnight at 4°C, this was followed by 45 min with Dynabeads (Invitrogen) to concentrate immunoglobulin complexes. Hsp90 pull downs were done as previously described (Taipale et al., 2012 (link)). For the identification of methylated peptides, proteins extracts were prepared in the presence of 160 μM of the demethylase inhibitor 5-carboxy-8-hydroxyquinoline (SML0067; Sigma-Aldrich).