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4 cut and fractionated by Superose-6 chromatography (25). Selected fractions were analyzed by immunoblotting with mAbs targeted against BARD1 (EE 6) (16), CstF-64 (9), or BRCA1 (MS110) (33). NE and fractions eluting from Superose-6 were immunoprecipitated with the anti-BARD1 polyclonal antibody, or the anti-CstF64 or BRCA1 mAbs, bound to protein A-Sepharose beads. Immunoprecipitations were carried out in buffer B at 4°C for 90 min, and washing was with buffer B. Aliquots of pellets and supematants were analyzed by SDS-PAGE and immunoblotting. The anti-BARD1 and CstF-64 antibodies did not cross-react with CstF-64 and BARD1, respectively, as judged by experiments with purified (BARD1-free) CstF and recombinant CST-BARD1 (22).
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Supported by National Institutes of Health (NIH) grant GM28983 to J.L.M. and an NIH Fogarty Fellowship to F.E.K. We thank Y. Takagaki for CstF plasmids and antibodies; R. Baer for BARD1 and BRCA1 encoding plasmids and anti-BARD1 antibodies; J. Chen and D. Livingston for the anti-BRCA1 antibody; R. Brent for yeast two-hybrid reagents; Y. Hirose for HeLa nuclear extract; and C. Prives, R. Baer, Y. Hirose, and R. Tacke for advice and discussions.
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