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

Abstract

<p>(A) ChIP assay showing the relative occupancy of the immunoprecipitated ERRα at the mouse <i>SCT</i> promoter in N42 cells after saline and ANGII treatment for 8 h in N-42 cells. Data was calculated using the equation: (Ct<sup>mock</sup>–Ct<sup>specific</sup>) 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 <i>in vitro</i> 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 <i>SCT</i> promoter in mouse hypothalamus after water deprivation and saline treatment (left) and ICV-ANGII injection in mice (1 and 4 h) (right).</p

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