Bile-acid-activated farnesoid X receptor regulates hydrogen sulfide production and hepatic microcirculation

Figure 3 Activation of FXR regulates CSE expression. A: FXR/RXR bind to CSE-IR1 of the CSE gene. EMSAs were performed to analyze binding of FXR/RXR to the putative IR1 sequence in the CSE gene. CSE-IR1, CSE-IR1_mutated and FXRE-IR1 probes, biotin-labeled, were used in this experiment. CSERE-IR1 probe was incubated with nuclear extracts from HepG2 cells not treated or treated with 6E-CDCA 10 &mgr;mol/L for 18 h. Competition experiments were performed using a 50-fold excess of unlabeled oligo or 1 &mgr;g of FXR antibody or 1 &mgr;g of RXR antibody. CSE-IR1_mutated and FXRE-IR1 probes were incubated with nuclear extracts from HepG2 stimulated cells; B: CSE-IR1 site binds FXR in the context of intact chromatin structures. ChIP experiments were performed with HepG2 cells. Chromatin was prepared and immunoprecipitated with antibodies directed against FXR and CD4. CD4 antibody was used as a negative control. Real-time PCR of the immunoprecipitated DNA by using the primer pairs indicated in Table 1. Data represent the mean ± SD of three experiments. ^aP < 0.05 versus not treated cells; C: Qualitative PCR of the immunoprecipitated DNA by using the primer pairs indicated in Table 1.