Fig. 3.
Biochemical assay of CYP2R1 vitamin D 25-hydroxylase enzyme activity. HEK 293 cells were transfected with the indicated expression plasmids for a period of 18–20 h. Thereafter, the medium was made 4.6 × 10-7 M in [4-14C]vitamin D3 and the incubation continued for an additional 96 h. Lipids were extracted from cells and medium into chloroform:methanol (2:1, vol/vol), and vitamin D metabolites and standards were separated by TLC on 150-Å silica gel plates (Whatman, catalog no. 4855–821) in a solvent system containing cyclohexane:ethyl acetate (3:2, vol/vol). After development, radioactivity was detected by PhosphorImager analysis, and the positions to which authentic vitamin D3 and 25-hydroxyvitamin D3 migrated on the plate were determined by staining with iodine.