Ab156575

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.

Western blot analysis was performed as previously described [36 (link)]. The following antibodies and reagents used: primary antibodies against Flag (#14793), PCNA (#13110), PKA C-α (#4782), CREB (#9197), pCREB (S133) (#9198), YAP (#14074) and pYAP (S127) (#13008) were purchased from Cell Signaling Technology, Inc. (diluted 1:1000; CST, MA, USA), a primary antibody against RAMP1 (#ab156575) was purchased from Abcam Plc (diluted 1:1000; Cambridge, UK), and a primary antibody against Gαi3 (#sc-365422) was purchased from Santa Cruz Biotechnology, Inc. (diluted 1:500; TX, USA); horseradish peroxidase (HRP)-linked anti-rabbit IgG (#7074) and HRP-linked anti-mouse IgG (#7076) secondary antibodies were purchased from CST (diluted 1:5000); and total protein-labelling No-Stain^™ reagent (#A44449) was purchased from Thermo Fisher Scientific.

NP tissue and cells were harvested using ReadyPrep Protein Extraction Kit (Bio‐Rad, 1632086). Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, 23225). Immunolabeling was detected using the Pierce ECL western blotting substrate (Pierce Biotechnology, 32132). The following antibodies and dilutions were used: CHSY1 antibody (1:1000) (Proteintech, 14420‐1‐AP), GAPDH (1:2000) (Abcam, ab9485); phospho‐CREB (1:1000) (Abcam, ab32096), CREB (1:1000) (Abcam, ab32515), RAMP1 (1:1000) (Abcam, ab156575).