5 years ago

Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

Marina Cecilia Muñoz, Diego Tomás Quiroga, Michael Bader, Valeria Burghi, Robson Augusto Souza Santos, Verónica Gabriela Piazza, Janaína Felix Braga, Érica Guilhen Mario, Yamila Belén Gándola, Natalia Cristina Fernández, Fernando Pablo Dominici

by Valeria Burghi, Natalia Cristina Fernández, Yamila Belén Gándola, Verónica Gabriela Piazza, Diego Tomás Quiroga, Érica Guilhen Mario, Janaína Felix Braga, Michael Bader, Robson Augusto Souza Santos, Fernando Pablo Dominici, Marina Cecilia Muñoz

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1–7) protective axis of renin–angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

Publisher URL: http://journals.plos.org/plosone/article

DOI: 10.1371/journal.pone.0183278

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