Figure 6.

Mas and B₂ receptors interact in physiological conditions. Immunoprecipitation of lysates from Wistar rat mesenteric vascular beds were performed using (A) anti-B₂R or (B) anti-MasR antibodies. Immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted (IB) using the indicated antibodies. These blots are representative of four different experiments with similar qualitative results. NS: non-specific, which refers to omission of the primary antibody in the coimmunoprecipitation assay (C) B₂R and MasR interaction measured by proximity ligation assay (PLA) in human tubulointerstitial and glomerular endothelial cells. Fixed cells were permeabilized with 0.2% Triton X-100 for 1 h, and then blocked in blocking buffer for 1 h. B₂R (anti-mouse) and MasR (anti-rabbit) antibodies were diluted in blocking solution, and incubated with the cells overnight at 4°C (A). PLA was conducted using Duolink® In Situ Orange Starter Kit Mouse/Rabbit (Sigma, St. Louis, MO, USA) following manufacturer´s instructions. Cells presented in image B were subjected to the same protocol but without the addition of the primary antibodies. Scale bar=15 µm