Convergence of Transforming Growth Factor-β and Vitamin D Signaling Pathways on SMAD Transcriptional Coactivators

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COS-1 cells were maintained in Dulbecco's modified Eagle's medium without phenol red, supplemented with fetal bovine serum (5%) treated with dextran-coated charcoal. The cells were transfected at 40 to 50% confluency in 10-cm petri dishes with a total of 20 μg of the indicated plasmids using calcium phosphate. All assays were done in the presence of 3 μg of pCH110 (Pharmacia), a β-galactosidase expression vector, as an internal control to normalize for variations in transfection efficiency. Cognate ligands were added to the medium 1 hour after transfection and at each exchange of medium. After 24-hour incubation with the calcium phosphate–precipitated DNA, the cells were washed with fresh medium and incubated for an additional 24 hours. Cell extracts were prepared by freezing and thawing and were assayed for CAT activity after normalization for β-galactosidase activity as described (9).

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GAL4 DNA-binding domain fusions were generated in pM, and VP16 fusions within pVP16. Interactions were tested in COS-1 cells. Activation of the CAT reporter–bearing GAL4-binding elements (17Mx5) was assayed in the presence or absence of 1,25(OH)₂D₃.

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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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.

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.