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Polyclonal antibodies were obtained by using the following bacterially expressed and purified GST fusion proteins as antigens: GST-hRad50-15A5, containing amino acids 211 through 575 of hRad50; GST-MM, containing amino acids 82 through 582 of hMre11; GST-NBS1, containing amino acids 12 through 754 of p95; GST-hRad51, containing full-length hRad51; and GST alone (for anti-GST mAb 8G11), to generate polyclonal or monoclonal antibodies (25). BRCA1 mAb's 6B4 and 17F8 were described in (18). BRCA1 mAb Ab-1 and rabbit α-hRad51 antibodies were from Oncogene Research Product (Cambridge, MA). Affinity-purified rabbit α-BRCA1 antibody (C-20) and α-HA antibody (Y-11) were from Santa Cruz Biotechnology (Santa Cruz, CA).
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1), cells were harvested. To obtain cells in M phase, we added nocodazole (0.4 μg/ml) to the culture medium for 10 hours before harvest.
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Constructs based on plasmid pcDNA3.1 (Invitrogen, San Diego, CA) were used for lipofectin-mediated transfection of HCC1937 cells with BRCA1 cDNA. Cells were harvested after 48 hours for immunoprecipitation and protein immunoblot analysis. Parallel cultures were treated with 0.1% MMS for 50 min, and surviving cells were counted after 8 days. The experiments were repeated at least three times, and the results were consistent.
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42
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We thank J. Petrini for rabbit antisera to hRad50, P. Garza and D. Jones for antibody production, and N. Ting and T. Boyer for critical reading. Supported by grants from NIH (CA 58183 and CA 30195), the McDermott endowment (W.H.L.), and the Susan G. Komen Foundation for Breast Cancer Research (P.L.C.).
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