Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type 1 Receptor Protein
The angiotensin II type 1 receptor (AT1R) mediates most hypertensive actions of angiotensin II. To understand the molecular regulation of the AT1R in normal physiology and pathophysiology, methods for sensitive and specific detection of AT1R protein are required. Here, we examined the specificity of a panel of putative AT1R 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 AT1R isoform, AT1A (AT1AKO), or with combined deficiency of both the AT1A and AT1B isoforms (AT1ABKO). For the 3 antibodies tested, Western blots of protein homogenates from wild-type kidneys yielded distinct bands with the expected size range for AT1R. In addition, these bands appeared identical in samples from mice lacking 1 or both murine AT1R isoforms. Additionally, the pattern of immunohistochemical staining in kidneys, liver, and adrenal glands of wild-type mice was very similar to that of AT1ABKO mice completely lacking all AT1R. We verified the absence of AT1R 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 AT1AKOs and AT1ABKOs. Finally, these antibodies failed to detect epitope-tagged AT1AR protein overexpressed in human embryonic kidney cells. We conclude that anti-AT1R antibodies available from commercial sources and commonly used in published studies exhibit nonspecific binding in mouse tissue that may lead to erroneous results.
- Received August 8, 2012.
- Revision received August 29, 2012.
- Accepted October 13, 2012.
- © 2012 American Heart Association, Inc.