Figure 5.

Binding of the VDR to promoter regions of TRPV6 and CaBP9k in lactating dams. A) mRNA and protein levels of the VDR in the duodenum of dams fed glucose, fructose, or starch diet for 6 wk. VDR mRNA levels were normalized to EF1α mRNA levels, while β-actin was used as loading and transfer control in Western blots. Relative mRNA levels were then normalized to those of dams fed starch. B) Schematic representation of the approximate location (solid rectangles) of the VDREs within the TRPV6 and CaBP9k promoters and of the primers (arrows) used for ChIP assays performed using anti-VDR antibodies. C, D) ChIP signals were detected by real-time RT-PCR using the primers to detect binding in the promoter regions of TRPV6 (C) and CaBP9k (D). Sonication of our samples generated DNA fragments of 250–500 bp. Because primers within 300–400 bp from the cis element of nuclear receptors are able to detect binding (15), primers designed as indicated in panel B should allow us to detect VDR interactions with the promoter regions of interest. ChIP signals were normalized to input signals. PCR product size was confirmed by electrophoresis on a 2% agarose gel. Only one band appears with the primer sets used (Supplemental Fig. S7). Bars are means ± se (n=3), with each n being analyzed twice and results averaged. Differences (P<0.05) among means within a region, indicated by differences in superscript letters, were analyzed by 1-way ANOVA LSD after an initial 2-way ANOVA showed significant diet or promoter region effects.