VDR•VDRE In Vitro Binding Assay.

A VDR expression plasmid was prepared by amplifying the VDR coding sequence (RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_000376.2","term_id":"63054843","term_text":"NM_000376.2"}}NM_000376.2), using LS180 cDNA as template, Herculase II Fusion DNA Polymerase (Agilent Technologies), and the primers listed in Supplemental Table 1. The amplified fragment was digested with HindIII and XhoI and ligated into the pcDNA3.1 expression plasmid (Life Technologies). Approximately 1,000,000 HEK293 cells were seeded into 100-mm plates. On reaching 70%–80% confluency, the cells were transfected with 60 μl Lipofectamine 2000 (Life Technologies), 1.5 µg of VDR-pcDNA3.1, and 1.5 µg RXRα-pSG5 (provided by Dr. Steven Kliewer, University of Texas Southwestern, Dallas, TX). Forty-eight hours later, nuclear proteins were extracted using the NucBuster Protein Extraction kit (Novagen; EMD Millipore, Billerica, MA), and protein concentrations were quantified using the bicinchoninic acid protein assay (Thermo Fisher Scientific, Rockford, IL).

The Universal EZ-TFA Chemiluminescent Transcription Factor Assay (EMD Millipore) was performed according to the manufacturer’s protocol. Briefly, 0.75 µg of HEK293 nuclear extract containing VDR and RXRα, 2 pmol biotinylated capture probe containing the VDRE consensus sequence from the rat osteocalcin gene (also known as bone γ-carboxyglutamate, official symbol Bglap; Markose et al., 1990; Demay et al., 1992), and transcription factor assay buffer were added to the wells of a streptavidin-coated microplate.

Competition for binding of VDR to the capture probe was assessed by adding one of the following unlabeled double-stranded oligonucleotides to the binding reaction: 1) consensus VDRE, 2) mutated consensus VDRE (Gutierrez et al., 2004), 3) SULT1C2 VDRE, or 4) mutated SULT1C2 VDRE. The competitors were added at 1.5- to 50-fold molar excess of the biotinylated capture probe. Background binding was determined by assessing VDR binding to a biotinylated mutated consensus VDRE probe. Capture probe, competitor probe, and negative control probe sequences are listed in Supplemental Table 1. Capture probe-bound VDR was detected using the primary antibody VDR (D-6) X (sc-13133X; Santa Cruz Biotechnology, CA) diluted 1:5000, followed by horseradish peroxidase-conjugated secondary antibody (1:500 dilution). Chemiluminescence was measured using kit reagents and a GloMax Luminometer. Data are expressed relative to VDR•RXR binding to the consensus VDRE capture probe in the absence of a competitor.