Bard1 h 300

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).

HEK293 cells were lysed in isotonic buffer with 1% Triton X-100. Lysate containing 400 μg of protein was incubated with 2 μg of antibody immobilized on protein G-Sepharose (Qiagen) over night at 4°C. The beads were washed with lysis buffer and proteins were eluted by heating at 95°C for 5 min in SDS loading buffer supplemented with 100 mM DTT.
Samples were subjected to SDS-PAGE and electro-blotted onto Immobilon-P (Millipore). Specific proteins were immuno-detected and visualized using enhanced chemiluminescence reagent (Amersham Biosciences) and X-ray film or GeneGnome chemiluminescence detection system (Syngene). The signal intensity was quantified using AlpfaEaseFC software (Alpha Innotech). For normalization of signals, Input or control signals from the same membrane were used. The antibodies used were BARD1 N19 (Santa Cruz), BARD1 H300 (Santa Cruz), TRF1 (Santa Cruz), TRF2 (Santa Cruz), TNKS (ThermoFisher Scientific), ABC biotin detection kit (ThermoFisher Scientific).