FIG. 3.

Vitamin D analog-induced interactions between GST-VDR and SRC-1/TIF2 family proteins in a GST pull-down assay. GST-VDR was expressed in E. coli and immobilized on glutathione-Sepharose beads. In vitro-translated SRC-1 and TIF2 labeled with [³⁵S]methionine were incubated with the beads in the absence of added ligand (−) or in the presence of 1α,25(OH)₂D₃, F₆-1α,25-(OH)₂D₃, ED-71, OCT, or 24R,25(OH)₂D₃ at a concentration of 10⁻⁸ M. The bound proteins were analyzed by SDS–7.5% PAGE and visualized by autoradiography. As a control, 1/10 of the amount of labeled SRC-1 (left panel) and TIF2 (right panel) included in the binding reactions are shown in the first lane. Representative GST pull-down assays and graphs corresponding to means ± standard deviations for triplicate independent experiments are shown. Note that any interaction of GST with these coactivators was not induced in the presence of ligands (data not shown).