for the untreated samples was obtained; this value was then subtracted from each of the Ct’s of all samples. These values represented the Ct’s. Each Ct was then entered into the equation: 2 − Ct . Averages were obtained for the treated and untreated groups, and norxacin results reported as percent of untreated values. In experiments involving low and high calcium treatment, low calcium treatment represented the “untreated” group in the calculations. Regulation of 1 .
OHase mRNA in human parathyroid cells Western Blot analysis of 1 OHase in kidney and parathyroid tissue. Immunoblot of rat kidney and human parathyroid tissue using an Daidzin antibody raised to a C-terminal peptide of the rat 1 OHase revealed a major band of the correct size . Protein degradation products are observed in the kidney sample, and a faint, non-speciﬁc band was detected at approximately 75, Da. The effect of cAMP or FGF-23 on 1 OHase expression was exam- ined in human parathyroid cells cultured in suspension. Cells were collected, centrifuged and resuspended in treatment medium consisting of serum-free medium with or without 2 mM dibutyryl cyclic AMP or ng/ml FGF-23 . After 1 h of treatment, the cells were centrifuged, medium discarded and RNAzol added to the cell pellet.
To examine the effect of calcium on 1 OHase expression in parathyroid cell monolayers, purchase ITMN-191 calcium-free, serum-free cell culture medium was supplemented with calcium chloride to obtain low calcium , normal calcium and high calcium treatment media. Phosphate concentration for the LC, NC and HC media was 97, 97 and 94 mM, respectively. Two to three days after plating, culture medium was removed from the cells and replaced with the appropriate treatment medium. After 24 h treatment, medium was removed and RNAzol added to the monolayers. The effect of the calcimimetic cinacalcet on 1 OHase expression in parathyroid cell monolayers was also examined. Monolayers were treated with serum-free medium containing ethanol or cinacalcet .
After 24 h treatment, medium was removed, and RNAzol added to the monolayers. order Decitabine Regulation of 1 . OHase activity in human parathyroid cells by calcium To determine the effect of calcium on 1 OHase activity , monolayers of human parathyroid cells were incubated for 24 h with low calcium or high calcium medium containing nM of 25D 3 . Duplicate samples of media were incubated under identical conditions, but without cells, to control for cross-reactivity of 25D 3 in the assay. Medium was collected and analyzed for 1-hydroxylated metabolites using the calcitriol radioimmunoassay from Immunodiagnostics Systems Ltd. , as previously described . The antibodies used in this assay recognize the 1-hydroxyl group and detects not only calcitriol, but all 1-hydroxylated spectrum metabolites, including calcitroic acid, the end-product of calcitriol metabolism by the 24-hydroxylase. Therefore, the assay provides a more accurate assessment of activity than simply measuring transient levels of calcitriol in the cultures.