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

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          Abstract

          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.

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          The renin-angiotensin system and cancer: old dog, new tricks.

          For cancers to develop, sustain and spread, the appropriation of key homeostatic physiological systems that influence cell growth, migration and death, as well as inflammation and the expansion of vascular networks are required. There is accumulating molecular and in vivo evidence to indicate that the expression and actions of the renin-angiotensin system (RAS) influence malignancy and also predict that RAS inhibitors, which are currently used to treat hypertension and cardiovascular disease, might augment cancer therapies. To appreciate this potential hegemony of the RAS in cancer, an expanded comprehension of the cellular actions of this system is needed, as well as a greater focus on translational and in vivo research.
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            A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons.

            In vertebrates, peripheral chemosensory neurons express large families of G protein-coupled receptors (GPCRs), reflecting the diversity and specificity of stimuli they detect. However, somatosensory neurons, which respond to chemical, thermal, or mechanical stimuli, are more broadly tuned. Here we describe a family of approximately 50 GPCRs related to Mas1, called mrgs, a subset of which is expressed in specific subpopulations of sensory neurons that detect painful stimuli. The expression patterns of mrgs thus reveal an unexpected degree of molecular diversity among nociceptive neurons. Some of these receptors can be specifically activated in heterologous cells by RFamide neuropeptides such as NPFF and NPAF, which are analgesic in vivo. Thus, mrgs may regulate nociceptor function and/or development, including the sensation or modulation of pain.
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              GLP-1 receptor activation indirectly reduces hepatic lipid accumulation but does not attenuate development of atherosclerosis in diabetic male ApoE(-/-) mice.

              Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce lipid accumulation in peripheral tissues, attenuating atherosclerosis and hepatic steatosis in preclinical studies. We examined whether GLP-1R activation decreases atherosclerosis progression in high-fat diet-fed male ApoE(-/-) mice after administration of streptozotocin and treatment with the long-acting GLP-1R agonist taspoglutide administered once monthly vs. metformin in the drinking water for 12 wk. Taspoglutide did not reduce plaque area or lipid content in the aortic arch or abdominal aorta, and no significant change in aortic macrophage accumulation was detected after taspoglutide or metformin. In contrast, hepatic triglyceride levels were significantly reduced in livers from taspoglutide-treated mice. Both peripheral and intracerebroventricular administration of exendin-4 rapidly decreased plasma triglyceride levels in fasted mice, and taspoglutide therapy in ApoE(-/-) mice modulated the expression of hepatic genes controlling fatty acid uptake and oxidation. We were unable to detect expression of the entire Glp1r coding sequence in macrophages isolated from ApoE(-/-), C57BL/6, and IL10(-/-) mice. Similarly, Glp1r mRNA transcripts were not detected in RNA from isolated murine hepatocytes. Using Western blotting and tissue extracts from Glp1r(+/+) and Glp1r(-/-) mice, and cells transfected with a tagged murine GLP-1R cDNA, we could not validate the sensitivity and specificity of three different GLP-1R antisera commonly used for the detection of GLP-1R protein. Taken together, these findings illustrate divergent actions of GLP-1R agonists on atherosclerosis progression and accumulation of ectopic lipid in ApoE(-/-) mice and highlight the importance of indirect GLP-1R actions for the control of hepatic lipid accumulation.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: SupervisionRole: Writing – original draft
                Role: InvestigationRole: MethodologyRole: Supervision
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: ResourcesRole: Supervision
                Role: ResourcesRole: SupervisionRole: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                16 August 2017
                2017
                : 12
                : 8
                : e0183278
                Affiliations
                [1 ] Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina
                [2 ] Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Investigaciones Farmacológicas (ININFA), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina
                [3 ] INCT-NanoBiofar, Department of Physiology and Biophysics, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
                [4 ] Max-Delbrück Center for Molecular Medicine, Berlin, Germany
                [5 ] Cardiology Institute of Rio Grande do Sul/University Foundation of Cardiology (IC/FUC), Porto Alegre, Rio Grande do Sul, Brazil
                George Washington University School of Medicine and Health Sciences, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-0198-9290
                Article
                PONE-D-17-19138
                10.1371/journal.pone.0183278
                5558983
                28813513
                b3313532-579e-41ce-8243-e52a724766cf
                © 2017 Burghi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 May 2017
                : 1 August 2017
                Page count
                Figures: 7, Tables: 0, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100005363, Universidad de Buenos Aires;
                Award ID: 2002013010028BA
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100005363, Universidad de Buenos Aires;
                Award ID: 20020110300041
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100006668, Fondo para la Investigación Científica y Tecnológica;
                Award ID: PICT 2012-0694
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100006668, Fondo para la Investigación Científica y Tecnológica;
                Award ID: PICT 2014-0362
                Award Recipient :
                This study was supported by Agencia Nacional de Promoción Científica y Tecnológica (grant number PICT-2014-0362 and grant number PICT 2012-0694) and Universidad de Buenos Aires (grant number UBA 2002013010028BA and grant number UBA 20020110300041).
                Categories
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                Research and Analysis Methods
                Histochemistry and Cytochemistry Techniques
                Immunohistochemistry Techniques
                Research and Analysis Methods
                Immunologic Techniques
                Immunohistochemistry Techniques
                Biology and Life Sciences
                Anatomy
                Renal System
                Kidneys
                Medicine and Health Sciences
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                Renal System
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