Evaluation of histone acetylation status at the OREs of AR in response to hypertonicity.
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Abstract
<p>(A) Schematic illustration of the mouse AR genomic DNA showing exon 1 and 2, promoter and upstream region. The region amplified by primer sets AR<sub>INTER</sub>, AR<sub>ORE</sub>, AR<sub>PP</sub> and AR<sub>EX2</sub> are indicated by arrows. Transcriptional start site is indicated by a bold arrow. The DNA probe for southern blotting analysis in the MNase digestion assay (ORE<sub>MNase</sub>) is indicated by a grey bar. (B) ChIP assays of AR promoter. ChIP assays were performed with antibodies against acetylated histone H3 or acetylated histone H4 using NIH-3T3 cells that were induced with hypertonicity for 0, 2, 6 and 10 h, respectively. DNA was analyzed by real-time PCR using AR<sub>ORE</sub> primer pair. The results are normalized to the ratio of immunoprecipitated DNA to total input DNA at time 0. Data are the mean ± SEM (n = 3). *, p<0.05 by one-way ANOVA test when compared to the value at time 0. (C) Quantitative analysis of AR transcriptions by RT-PCR. NIH-3T3 cells were incubated in hypertonic medium for 0, 3, 6, 8 and 15 h. Total RNA was prepared. Relative expression level of AR was determined. The expression level of β-actin was used to normalize the AR expression. Results were expressed as fold change relative to the expression level at time 0. Data are the mean ± SEM (n = 3).</p
