Mab052

Total protein (100 μg) from the indicated NEs was immunoprecipitated as described previously (7 (link), 63 (link)). Immunoprecipitations (IPs) were performed with antibodies against CstF-50 (Bethyl), Ub (P4D1; Santa Cruz Biotechnology), HA (conjugated beads from Sigma), and p97 (Bethyl). Western blot analysis was done with antibodies against histones (MAB052; Millipore), RNAP IIO (H5; Covance), BRCA1 (sc-1021; Santa Cruz Biotechnology), BARD1 (H-300; Santa Cruz Biotechnology), Ub-H₂A (Millipore), Ub-H₂B (Millipore), and Topo II (Santa Cruz).

Cells were isolated, suspended in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Roche, NY, USA), triturated and centrifuged at 10,000 g for 15 minutes at 4°C. 1–10 μg of total protein was separated on 10% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane and probed with various primary antibodies, followed by horseradish-peroxidase-conjugated secondary antibody (1,:5000–10,000, for mouse or rabbit primaries respectively, Invitrogen), and visualized using ECL chemiluminescence (Pierce, IL, USA). Primary antibodies used in this study are GIG (1:150, a generous gift from Dr. Pascale Bomont), HISH3, (1:500, Millipore, MAB052), PRPH (1:5,000, kindly provided by Dr. Ron Liem).