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COS-1 cells were transfected with the indicated plasmids, lysed in TNE buffer [10 mM Tris-HCl (pH 7.8), 1% NP-40, 0.15 M NaCl, 1 mM EDTA], and immunoprecipitated with monoclonal antibody to FLAG (IBI; Eastman Kodak). Interacting proteins were separated by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (Bio-Rad), and then detected with antibody to VDR and antibody to rabbit immunoglobulin G conjugated with alkaline phosphatase (Promega).
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46
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35S]methionine-labeled proteins. Bound proteins were eluted and analyzed by SDS-PAGE.
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35S]methionine with the reticulocyte lysate system (Promega).
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2, 0.5% (v/v) NP-40] and incubated on ice for 15 min, then centrifuged again for 5 min at 500g. The sedimented nuclear fraction was resuspended in TNE buffer [10 mM tris-HCl (pH 7.8), 1% NP-40, 0.15 M NaCl, 1 mM EDTA], incubated for 30 min on ice, and centrifuged. The supernatant was used as nuclear extract for the experiments.
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q. Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. X indicates any residue.
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56
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0344748599
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note
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We thank P. Chambon for critical reading of the manuscript and for providing nuclear receptor expression vectors, H. Gronemeyer for providing TIF2 expression vectors, R. H. Goodman for the CBP cDNA, and S. Hanazawa and A. Takeshita for helpful discussion.
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