Ketoconazole k1003

25(OH)D₃ (BML-DM-100-0001) and 1,25(OH)₂D₃ (BML-DM200-0050) were from Enzo Life Sciences, Inc., Ann Arbor, MI. Stock solutions of 2.5 mM 25(OH)D₃ and 2.4 mM 1,25(OH)₂D₃ were prepared in anhydrous (≥99.5%) ethanol and stored at −80°C. To determine 1,25(OH)₂D₃ in the supernatants we used the 1,25-Dihydroxy Vitamin D EIA kit (AC-62F1) from IDS, Tyne and Wear, UK according to the manufacturer's instructions. Antibodies used included anti-VDR (D-6) and anti-CD3ζ (6B10.2) from Santa Cruz Biotechnology, Santa Cruz, CA, anti-p53 (9282) from Cell Signaling Technology, Danvers, MA, anti-GAPDH from (ab9485) from Abcam, Cambridge, MA and HRP-rabbit anti-mouse Ig (P0260) from DAKO, Glostrup, Denmark. Cycloheximide ready made solution 100 mg/ml in DMSO (C4859), phorbol 12-myristate 13-acetate (PMA, P8139), ionomycin (I0634), monensin (M5273), leptomycin B (LMB) (L2913) and ketoconazole (K1003) were from Sigma-Aldrich, St. Louis, MO. A fresh solution of ketoconazole 20 mg/ml in anhydrous ethanol was prepared before each experiment. The proteasome inhibitors lactacystin (426100) and MG-132 (474788) were from Merck Millipore, Nottingham, UK.

25(OH)D₃ (BML-DM100-0001) and 1,25(OH)₂D₃ (BML-DM200-0050) were from Enzo Life Sciences, Inc., Ann Arbor, MI. Stock solutions of 2.5 mM 25(OH)D₃ and 2.4 mM 1,25(OH)₂D₃ were prepared in anhydrous (≥99.5%) ethanol and stored at −80°C. To determine 1,25(OH)₂D₃ in the medium we used the 1,25-Dihydroxy Vitamin D EIA kit (AC-62F1) from IDS, Tyne and Wear, UK according to the manufacturer’s instructions. DBP (A50674H) and albumin (A8763) purified from human serum were from Meridian Life Sciences and Sigma-Aldrich, respectively. Actin (A2522), arachidonic acid (A9673) and ketoconazole (K1003) were from Sigma-Aldrich. Serum and central lymph from mini-pigs were provided by the Department for Experimental Medicine, University of Copenhagen, Denmark.