Fig. 4

FXR binds to the FXRE/DR2 in miR-122 promoter region. After treatment with the FXR agonist GW4064 (5 μM) for 48 h, Huh7 cells were harvested for EMSA or ChIP assays. a The sequences of DR2 probe and mutant (Mut) DR2 probe are shown. The DR2 element is in bold, and the mutated bases are underlined. b EMSA analysis of the binding of GW4064-treated Huh7 cell nuclear proteins to the FXRE/DR2. The reactions were analyzed by electrophoresis in a nondenaturing 4 % polyacrylamide gel, followed by autoradiography. The cold DR2 or Mut DR2 probe was used at 100× excess concentrations over the labeled probe in competition experiments. The antibody directed against FXR (anti-FXR) was used in supershift assays, taking IgG as a negative control. c ChIP assays were performed using chromatin isolated from GW4064-treated Huh7 cells. Anti-FXR was used for immunoprecipitation of the chromatin DNA fragment, taking IgG as a negative control. The precipitated DNA was extracted and amplified by PCR using primers spanning the DR2 element. The input (total DNA extract) was used as positive PCR control. No antibody (no anti-FXR and IgG in the reaction) was used as mock control