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The heterodimer associates with vitamin D-responsive elements in the promoter of vitamin D target genes such as 24-hydroxylase (The transformed PZ-HPV-7 cell line (CRL-2221) was obtained from ATCC (Manassas, VA, USA).The cell line was derived from epithelial cells of the peripheral zone of the normal prostate tissue by transfecting with HPV18 DNA.
The comparative threshold cycle values were used to determine the relative m RNA expression levels as described previously (Munetsuna si RNA: 1) 5′-AAAGAAUUUGGCUCUGGGAACUGGG-3′ and 5′-CCCAGUUCCCAGAGCCAAAUUCUUU-3′, 2) 5′-UAGGAUCUGGGCCAAAGCCAUUUGC-3′ and 5′-GCAAAUGGCUUUGGCCCAGAUCCUA-3′, 3) 5′-AAUGCAAACAUCUGGUCCCAGUCUC-3′ and 5′-GAGACUGGGACCAGAUGUUUGCAUU-3′; si RNA: 1) 5′-UUGCCAAACACUUCGAGCACAAGGG-3′ and 5′-CCCUUGUGCUCGAAGUGUUUGGCAA-3′, 2) 5′-UAGCAUUGAAGUGAAAGCCAGUGGC-3′ and 5′-GCCACUGGCUUUCACUUCAAUGCUA-3′, 3) 5′-UUUGGAUGCUGUAACUGACCAGGUC-3′ and 5′-GACCUGGUCAGUUACAGCAUCCAAA-3′.
Each set of duplex si RNAs was transfected into PZ-HPV-7 cells for 24 h using Lipofectamine RNAi Max according to the manufacturer's instructions before treating with 19-nor-vitamin DProteins were separated on a 10% SDS–PAGE and transferred to nitrocellulose transfer membrane (GE Healthcare, Buckinghamshire, UK).
The conditions of liquid chromatography (LC) were as follows: column, reverse-phase ODS column (μBondapak C18, 5 μm; Waters, Milford, MA, USA; 6×150 mm); mobile phase, 80% methanol aqueous solution per 25 min; flow rate, 1.0 ml/min; and u.v.
detection, 254 nm (Urushino for 0–90 min, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized in 0.1% Tween-20/PBS, and blocked with 1% BSA.
is not suitable as a therapeutic agent for cancer treatment.
Accordingly, the analogs that are less calcemic but exhibit potent anti-proliferative activity have potential as therapeutic agents.
Among the analogs of 1α,25(OH) are mediated by the vitamin D receptor (VDR).
On binding of the hormone to the receptor, VDR heterodimerizes with retinoid X receptor (RXR) and translocates into the nucleus (Racz & Barsony 1999, Prufer & Barsony 2002).
Cells were washed and incubated with antihuman VDR rat monoclonal antibody (Abcam, Cambridge, UK) for 2 h at room temperature.
Then, cells were washed and incubated with Alexa-555-labeled anti-rat secondary antibody (Invitrogen) for 2 h.