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

Figure 3.

Figure 3

Effect of VDR ligands on tyrosine phosphorylation and interaction of VDR and c-Src, and CYP7A1 mRNA expression levels in human primary hepatocytes. A, IP assay of tyrosine phosphorylation of VDR in the plasma membrane. Primary human hepatocytes were treated with vehicle (Veh, EtOH),1α, 25(OH)2-D3 (50 nm), LCA (10 μm), LCA-acetate (10 μm), CDCA (20 μm), or CA (20 μm) for 6 h. A rabbit anti-VDR antibody was used to immunoprecipitate VDR from cell membrane extracts (300 μg). A mouse antiphosphotyrosine (Anti-p-Tyr) was used to detect phosphotyrosines in VDR. Cell extract (10%) was set aside as input. B, Co-IP assay of VDR interaction with c-Src. Primary human hepatocytes were treated with Veh, 1α,25(OH)2-D3 (50 nm), or LCA-acetate (10 μm) for 6 h. Cell membrane extracts (300 μg) were treated with a mouse anti-VDR antibody, and immunoprecipitates were used for immunoblot analysis of coprecipitated c-Src using rabbit anti-c-Src and anti-p-Y416/Y419-Src antibodies. Mouse nonimmune IgG was used as a negative control. Cell extract (10%) was set aside as input. C, Effect of a c-Src inhibitor on tyrosine phosphorylation of VDR in the plasma membrane. Primary human hepatocytes were treated with Veh, 1α,25(OH)2-D3 (50 nm), LCA-acetate (10 μm), and/or the c-Src inhibitor AZD0530 (AZD) (5 μm) for 6 h. A rabbit anti-VDR antibody was used to immunoprecipitate VDR from cell membrane extracts (300 μg). A mouse antiphosphotyrosine (Anti-p-Tyr) was used to detect phosphotyrosines in VDR. A mouse anti-VDR was used to detect immunoprecipitated VDR. Cell extract (10%) was set aside as input. D, Q-RT PCR assay of the effects of a c-Src inhibitor on CYP7A1 (left panel) and CYP24A1 (right panel) mRNA expression in primary human hepatocytes. Primary human hepatocytes were treated with Veh, 1α,25(OH)2-D3 (50 nm), LCA-acetate (10 μm), and/or AZD (5 μm) for 16 h. #, Statistically significant difference (P < 0.05; n = 3), AZD-treated vs. vehicle control, or AZD and 1α,25(OH)2-D3 (50 nm) or LCA-acetate (10 μm) treated vs. 1α,25(OH)2-D3 (50 nm) or LCA-acetate (10 μm). *, Statistically significant difference (P < 0.05; n = 3), 1α,25(OH)2-D3 (50 nm) or LCA-acetate (10 μm) treated vs. vehicle control. Data represent one of three separate experiments using primary human hepatocytes from different liver donors (HH1479, HH1483, and HH1493).