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+ RNA (27). The cDNA fragments were subsequently inserted into the Hind III site of the mammalian expression vector pcDNA3 (Invitrogen). The reporter plasmid 17M2-G-lacZ was constructed by inserting yeast GAL4 upstream activating sequence x2 and β-globin promoter into a multicloning site of the Basic expression vector (Clontech). The ligand-binding domain of VDR [VDR(DEF)] fused to the GAL4 DNA-binding domain [GAL4-VDR(DEF)] (11) was used to detect ligand-induced AF-2 function. COS-1 cells cultured with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum were transiently transfected with 0.5 μg of GAL4-VDR(DEF) expression vector, 1 μg of 17M2-G-lacZ, 0.2 μg each of the ADX and ADR expression vectors (12), and 0.1 μg of expression cDNA library in OPTI-MEM using lipofection agent (GIBCO BRL).
-
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56
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9844266465
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note
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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.
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58
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9844261390
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We thank P. Chambon for critical reading of the manuscript; T. Yoshizawa, Y. Masuhiro, S. Takeda, K. Suzuki, F. Ichikawa, N. Kubota, N. Ohishi, K. Kumaki, and A. Murayama for technical assistance; Y. Nogi, T. Sakaki, and T. Matsumoto for helpful discussion and suggestions; Y. Yabusaki for the ADX and ADR cDNA plasmids; and Chugai Pharmaceuticals for vitamin D-related compounds.
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