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. 2010 Apr 6;24(6):1151–1164. doi: 10.1210/me.2009-0482

Figure 9.

Figure 9

ChIP assay of nuclear receptors and cofactors association with human CYP7A1 promoter (A) and CYP24A1 promoter (B). Primary human hepatocytes were pretreated with U0126 (U) (20 μm) for 1 h followed by treatment with 1α,25(OH)2-D3 (50 nm) or LCA-acetate (10 μm) for 16 h. Rabbit anti-VDR, anti-RXRα, and anti-HNF4α antibodies, and goat anti-PGC-1α, anti-NCoR-1, and anti-SMRT antibodies were used to immunoprecipitate the chromatin fragments. Nonimmune IgG was used as a negative control. Cell extract (5%) was set aside as input. A, Taqman primer set probes were designed for Q-PCR to detect human CYP7A1 promoter region containing BARE-II and BARE-II (HNF4α and VDR/RXRα binding site) and intron 5 (+9127 to +9193) (a negative control), as described in Materials and Methods. B, Tagman primer set probes were designed for Q-PCR to detect VDRE in the proximal region of human CYP24A1 promoter. Data were pooled from assays using three donor hepatocytes (HH1460, HH1479, and HH1483). *, Statistically significant difference (P < 0.05; n = 3) vs. vehicle (Veh) control. #, Statistically significant difference (P < 0.05; n = 3) vs. LCA-acetate or 1α, 25(OH)2-D3 treatment.