Donkey anti mouse igg hrp conjugate

Antibodies were from the following sources: Anti-Nr2f2 mouse monoclonal (Nr2f2-Ab1, Abcam, 1:2000 dilution), anti-CoupTF II (Nr2f2-Ab2, Cell signaling, 1:500 dilution), anti-Fog2 (GeneTex, 1:1000 dilution), donkey anti-mouse IgG-HRP conjugate (SC-2020, 1:5000 dilution), anti-rabbit IgG-HRP conjugate (Cell Signaling, 1: 5000 dilution). Samples were homogenized in ice-cold RIPA lysis buffer with protease inhibitor cocktail (Pierce). Immuno-blotting for Nr2f2 was carried out using tissue lysates from Nr2f2^mutant and wild-type hypertensive rat homogenates. In brief, forty microgram of proteins were boiled with Laemmli sample buffer (Bio-Rad) for 5 minutes at 95° C. Protein samples were resolved using 4–15% Criterion Tris-HCl Gel (Bio-Rad) at room temperature, and transferred on to PVDF membrane (Millipore), blocked with 5% fat free milk powder in PBST and incubated with anti-Nr2f2 and secondary antibodies conjugated with horseradish peroxidase. Immunoreactivity was detected by autoradiography using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Full-length blots are shown in Supplementary Figure 1.