PERSONAL DE APOYO
MUÑOZ marina Cecilia
artículos
Título:
Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies
Autor/es:
BURGHI V; FERNANDEZ NC; GÁNDOLA Y; PIAZZA VG; QUIROGA DT; GUILHEN MARIO E; FELIX BRAGA J; BADER M; SANTOS RAS; DOMINICI FP; MUÑOZ MC
Revista:
PLOS ONE
Editorial:
PUBLIC LIBRARY SCIENCE
Referencias:
Lugar: San Francisco; Año: 2017 vol. 12 p. 1 - 19
ISSN:
1932-6203
Resumen:
Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediatingthe 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 tissuedistribution and expression levels constitutes a critical knowledge to fully understanding itsfunction. Commercially available antibodies have been widely employed for MasR proteinlocalization and quantification, but they have not been adequately validated. In this study, wecarried on an exhaustive evaluation of four commercial MasR antibodies, following previouslyestablished criteria. Western Blotting (WB) and immunohistochemistry studies starting fromhearts and kidneys from wild type (WT) mice revealed that antibodies raised against differentMasR domains yielded different patterns of reactivity. Furthermore, staining patterns appearedidentical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chainreaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcriptswere undetectable in either heart or kidney from this animal model. In addition, we evaluatedthe ability of the antibodies to detect the human c-myc-tagged MasR overexpressed inhuman embryonic kidney cells. Three antibodies were capable of detecting the MasR either byWB 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 athigh expression levels observed in a transfected cell line, they failed to detect this receptor inmice tissues at physiological expression levels. As a consequence, validated antibodies thatcan recognize and detect the MasR at physiological levels are still lacking.