Figure 5. Effect of saline and ANGII on the binding of ERRα to the mouse SCT promoter.

(A) ChIP assay showing the relative occupancy of the immunoprecipitated ERRα at the mouse SCT promoter in N42 cells after saline and ANGII treatment for 8 h in N-42 cells. Data was calculated using the equation: (Ct^mock–Ct^specific) and normalized with Input, which was defined as 1.00. A mock immunoprecipitation using rabbit anti-IgG and without using antibody were carried out as negative control. Data represent the mean ± SEM of three experiments.*p<0.05; **p<0.001, compared with the control. (B) EMSA using the ERE-half site as the oligo. Left: Nuclear extract of mouse hypothalamus (10 µg), pcDNA (control), and in vitro translated ERRα protein were pre-incubated with the oligo for 15 minutes at room temperature. Arrows indicate specific protein-DNA complexes that contain ERRα (Complex I) and unknown protein (complex II). Middle: Hypothalamic nuclear extract of control or water deprived or saline-drank mice were used (n = 4/group) in EMSA. Right: Supershift assay of ERRα proteins. Antibodies (2 µg) specific for ERRα and AP1 (Santa Cruz) proteins were pre-incubated with the nuclear extract (10 µg) for 20 min at room temperature. Arrows indicate specific protein–DNA complexes that contain ERRα (Complex I), non-specific complex (Complexes II) and ERRα supershift (SS). (C) ChIP assay showing the in vivo binding of ERRα on mouse SCT promoter in mouse hypothalamus after water deprivation and saline treatment (left) and ICV-ANGII injection in mice (1 and 4 h) (right).