FIGURE 4

Different half-lives of VDR-ligand conformations. Limited protease digestion assays were performed by preincubating in vitro translated [³⁵S]-labeled VDR together with nonlabeled in vitro translated RXR and the rat ANF DR3-type VDRE and saturating concentrations (10 M) of 1,25(OH)₂D₃, its analogs or solvent (as a control). The samples were incubated for indicated times with trypsin and were then electrophoresed through 15% sodium dodecyl sulfate–polyacrylamide gels. The amount of ligand-stabilized VDR conformations (sum of c1_LPD and c3_LPD) in relation to VDR input was quantitated by phosphorimaging. Representative experiments are shown for all four VDR agonists and solvent control. Data points represent the mean of triplicates and the bars indicate SD. The half-lives (t_1/2) were determined from the respective time-course curves.