Protein Expression Analysis in the PVN

Western blotting was used to measure the levels of protein expression of AT1R (ab124734), IMD (ab198273), RAMP1/2/3 ((ab-156575, ab-198276, ab-197372), NOX2 (ab129068) and NOX4 (ab133303), antibodies from Abcam, Burlingame, CA, USA) and ERK (T-ERK (16443-1-AP) antibody from Proteintech Group, Inc. Rosemont, IL, USA; P-ERK (AP1015) and CRLR (DF10203) antibodies from Affinity Biosciences. Cincinnati, OH, USA) activation in the PVN. The methods were as described previously [11 (link),19 (link)]. Protein loading was controlled by probing all blots with GAPDH antibody (antibody from Bioworld Technology, Louis Park, MN, USA) and the protein intensity was normalized to that of GAPDH.