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FIG. 2

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Different receptors preferentially interact with different domains of SMRT in an in vitro assay. Different nuclear hormone receptors were synthesized as radiolabeled proteins by transcription and translation in vitro and were tested for the ability to bind to nonrecombinant GST or to different GST-SMRT fusions immobilized on a glutathione-agarose matrix (as indicated above the panels). The assays were performed in the presence (+) or absence (−) of 100 nM cognate hormone. After the matrix was washed, the receptors remaining bound to the immobilized GST fusion proteins were eluted, resolved by SDS-PAGE, and visualized by PhosphorImager analysis. The amount of a radiolabeled receptor bound to a GST fusion polypeptide was also quantified and is presented numerically below each panel as a percentage of the total radiolabeled receptor (input) used in each binding reaction. The receptors tested were T3Rα, RARα, RXRα, and VDR. The ability of VDR to bind to an immobilized GST-RXR construct was also tested as a positive control (first two lanes of the bottom panel) to confirm the functionality of the in vitro-synthesized receptor. RA, retinoic acid.

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