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

Figure 7.

Figure 7

VDR ligand-dependent interaction of VDR with HNF4α, RXRα, coactivators, and corepressors. A, Effect of an ERK1/2 pathway inhibitor on VDR and HNF4α interaction. Primary human hepatocytes were pretreated with U (20 μm) for 1 h followed by treatment with 1α,25(OH)2-D3 (50 nm) or LCA-acetate (10 μm) for 6 h. An anti-HNF4α or anti-VDR antibody was used to immunoprecipitate VDR and HNF4α in the cell extracts (300 μg). Coimmunoprecipitated VDR and HNF4α were detected by immunoblot assay. B, Mammalian two-hybrid assay of VDR and HNF4α interaction and effect of coactivators and corepressors. The Gal4 reporter construct p5XUAS-TK-Luc (0.2 μg) was transfected with 0.1 μg of Gal4-VDR with VP16 empty vector, pcDNA3.1, VP16-HNF4α, PGC-1α, SRC-1, NCoR-1, and/or SMRT, as indicated, into HEK293 cells. After transfected for 24 h, cells were treated with vehicle (EtOH), 1α,25(OH)2-D3 (10 nm), or LCA-acetate (5 μm) for 16 h before harvesting. The experiment was done in duplicate, and the same experiment was repeated three times. *, Statistically significant difference (P < 0.05, n = 3) Gal4-VDR cotransfected with PGC-1α, SRC-1, or VP16-HNF4α vs. Gal4-VDR cotransfected with VP16 empty vector or pcDNA3.1. #, Statistically significant difference (P < 0.05; n = 3) Gal4-VDR and VP16-HNF4α cotransfected with PGC-1α, SRC-1, SMRT, or NCo-R-1 vs. Gal4-VDR and VP16-HNF4α cotransfected with pcDNA3.1. C, Mammalian two-hybrid assays of effect of RXRα on VDR interaction with NCoR-1, SMRT, or SRC-1. The GAL4 reporter construct p5XUAS-TK-Luc (0.2 μg) was cotransfected with 0.1 μg Gal4-VDR, RXRα, VP16 empty vector, pcDNA3.1, VP16-NCoR-1, VP16-SMRT, and/or VP16-SRC-1, as indicated, into HEK293 cells. After transfection for 24 h, cells were treated with vehicle (Veh, EtOH), 1α,25(OH)2-D3 (10 nm), or LCA-acetate (5 μm) for 16 h before harvesting. *, Statistically significant difference (P < 0.05, n = 3) Gal4-VDR cotransfected with VP16-SRC-1 vs. Gal4-VDR cotransfected with VP16 empty vector. #, Statistically significant difference (P < 0.05, n = 3), Gal4-VDR and RXRα cotransfected with VP16-NCoR-1, VP16-SMRT, or VP16-SRC-1 vs. Gal4-VDR and RXRα cotransfected with VP16 empty vector. All transfection experiments were done in duplicate, and the same experiment was repeated three times. RLU, Relative light units; U, U0126.