OSE cells (n = 3 patients) were plated at 100,000 cells/well in a 6-well plate. Twenty-four hours post plating, cells were treated with 0 (control) or 5 nM calcitriol (biologically active form of VD; Sigma-Aldrich) supplementation in the culture medium for 72 hrs. Cells were harvested for Western blot, as previously described [50 (link)]. Briefly, total protein was extracted using a RIPA buffer containing 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and 25 mM Tris-HCl (pH 7.6), electrophoresed on 4–12% Bis-Tris gel, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) nonfat milk in 10 mM Tris-buffered saline (pH 8.0), and then incubated with mouse anti-human VDR (1:250; sc-13133; Santa Cruz Biotechnology, Inc.) or α-Tubulin (loading control; 1:24,000; T6074; Sigma-Aldrich) antibody at 4°C overnight. The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, Burlingame, CA, USA). Images developed on the film were scanned using the Konica Minolta Bizhub C368 (Konica Minolta, Wayne, NJ, USA). Densitometry analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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