title: Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein.
abstract: The angiotensin II type 1 receptor (AT(1)R) mediates most hypertensive actions of angiotensin II. To understand the molecular regulation of the AT(1)R in normal physiology and pathophysiology, methods for sensitive and specific detection of AT(1)R protein are required. Here, we examined the specificity of a panel of putative AT(1)R antibodies that are commonly used by investigators in the field. For these studies, we carried out Western blotting and immunohistochemistry with kidney tissue from wild-type mice and genetically modified mice lacking the major murine AT(1)R isoform, AT(1A) (AT(1A)KO), or with combined deficiency of both the AT(1A) and AT(1B) isoforms (AT(1AB)KO). For the 3 antibodies tested, Western blots of protein homogenates from wild-type kidneys yielded distinct bands with the expected size range for AT(1)R. In addition, these bands appeared identical in samples from mice lacking 1 or both murine AT(1)R isoforms. Additionally, the pattern of immunohistochemical staining in kidneys, liver, and adrenal glands of wild-type mice was very similar to that of AT(1AB)KO mice completely lacking all AT(1)R. We verified the absence of AT(1)R subtypes in each mouse line by the following: (1) quantitative polymerase chain reaction documenting the absence of mRNA species, and (2) functionally by assessing angiotensin II-dependent vasoconstriction, which was substantially blunted in both AT(1A)KOs and AT(1AB)KOs. Finally, these antibodies failed to detect epitope-tagged AT(1A)R protein overexpressed in human embryonic kidney cells. We conclude that anti-AT(1)R antibodies available from commercial sources and commonly used in published studies exhibit nonspecific binding in mouse tissue that may lead to erroneous results.
authors.affiliation: Division of Nephrology, Department of Medicine, Duke University Medical Center, Durham, NC, USA. marcela.herrera@duke.edu
authors.forename: Marcela
authors.initials: M
authors.lastname: Herrera
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Sparks
authors.forename: Adolfo R
authors.initials: AR
authors.lastname: Alfonso-Pecchio
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Harrison-Bernard
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Coffman
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D007150:Immunohistochemistry; D007668:Kidney / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D044140:Receptor, Angiotensin, Type 1 / Q000276:immunology* / Q000378:metabolism
chemical_list: D044140:Receptor, Angiotensin, Type 1