Anti at2r

The PVN tissue was homogenized in a lysis buffer, and the protein concentration in the supernatant was measured with the BCA protein assay Kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 5% nonfat dry milk and then incubated using a primary antibody at 4°C overnight. The following primary antibodies used in this study have been verified in previous published literatures: anti-renin (sc-22752) [20 (link)], anti-AT1-R (sc-1173) [21 (link),22 (link)], anti-AT2-R (sc-9040) [23 (link)], anti-ACE-1 (sc-20791) [22 (link)], anti-ACE-2 (sc-20998) [24 (link)], anti-TNF-α (sc-1350) [25 (link)], anti-IL-1β (sc-7884) [25 (link)], anti-IL-10 (sc-57245) [26 (link)], and anti- β-actin (sc-47778) [22 (link)] (all antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA). After three washings, the membranes were incubated with horseradish peroxidase-conjugated second antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) for 1 h at room temperature. The signal was visualized using the enhanced chemiluminescence (ECL) detection system (Amersham), and the densities of the immunobands were quantitated using NIH ImageJ software (Bethesda, MD, USA). All data were corrected by β-actin.

Total protein was extracted after homogenizing the rat renal cortex, and the concentration was determined with a bicinchoninic acid protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes, which were incubated with the primary antibodies rabbit anti-β₁-AR (1:200), anti-β₂-AR (1:200), anti-β₃-AR (1:200), anti-α_1A-AR (1:200), anti-α_1B-AR (1:200), anti-α_1D-AR (1:200), anti-AT₁R (1:200), anti-AT₂R (1:200), and anti-GAPDH (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight, followed by incubation with goat anti-rabbit fluorescent (IRDye-conjugated) secondary antibodies (1:10,000; Rockland Immunochemicals, Gilbertsville, PA, USA) for 2 h at room temperature. The images were quantified by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Levels of proteins were normalized to that of GAPDH.