Lack of specificity for AT1 angiotensin receptor antibodies in Western blot analysis

Abstract

The angiotensin II type 1 receptor (AT1R) mediates the classical actions of angiotensin II (Ang II). In rodents, there are two AT1R, AT1a and AT1b, which are highly homologous (>90% at the amino acid level) and encoded by different genes. Detection of AT1R protein is important for a broad range of experimental approaches to understand molecular mechanisms of disease. While several anti AT1R antibodies are available commercially and used by investigators for these purposes, we have had concerns about their specificity. To address this, we examined the specificity of a panel of commercial anti AT1R antibodies (Santa Cruz 1173, AbCam 18801, Alomone AAR 011) for Western blot analysis. For these studies, we used kidney tissue from wild type mice and mice with genetic deficiencies of AT1a receptors (AT1a KO) or combined deficiency of AT1a and AT1b receptors (AT1ab KO), which completely lack AT1. For all three antibodies tested, a ~42 kDa band consistent with the predicted size of the AT1 was detected in tissues from WT mice. However, identical bands were also detected in tissues from the AT1a KO and AT1ab KO mice (n=3). Along with genotyping, we verified the absence of mRNA for the AT1a and AT1b receptors in KO mice and confirmed the absence of pressor responses to Ang II. We conclude that many of the available anti‐AT1R antibodies exhibit non‐specific binding that may lead to anomalous results.