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Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type 1 Receptor Protein

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.112.203679Hypertension. 2013;61:253–258

Abstract

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.

Introduction

The type 1 angiotensin receptor (AT1R) is a key component of the renin–angiotensin system. AT1R mediates most of the classically recognized actions of angiotensin II. Activation of AT1Rs stimulates vascular contractility and renal sodium retention14 playing a crucial role in regulating blood pressure in health and disease. In addition to these hemodynamic effects, the AT1R also mediates cell proliferation, fibrosis, and end organ damage.57 Because of the key role of these receptors in physiology and pathophysiology, accurate detection of the AT1R protein is paramount for investigations aimed at understanding molecular mechanisms of health and disease. Although several AT1R antibodies are available commercially we, and others,8 had major concerns about their specificity.

Humans have only 1 AT1R isoform; however, 2 isoforms exist in rodents, termed AT1A and AT1B. These receptor subtypes are products of 2 different genes: Agtr1a is located on mouse chromosome 13 (17 in rats), and Agtr1b on mouse chromosome 3 (2 in rats). They both encode a 375-amino acid protein with a predicted molecular weight of 42 kDa.911 They share 94% identity and are indistinguishable pharmacologically. Based on bioinformatics predictions (http://www.cbs.dtu.dk/services/NetNGlyc/), these receptors are presumed to undergo post-translational glycosylation. Accordingly, using a plasmid expressing the AT1R tagged to a myc epitope, Deslauriers and colleagues reported massive AT1R glycosylation in transfected COS-7 cells.12 In that study, the molecular size of the glycosylated AT1R form was estimated to be ≈100 to 150 kDa. Although the degree of glycosylation of proteins is a tissue-specific process, it is difficult to predict the molecular mass of these receptors under different tissues or experimental conditions, and published data clarifying this issue are lacking.

During the last decade, anti-AT1R antibodies have been widely used in scientific reports related to AT1R signaling and functions. However, their specificity has not been thoroughly investigated in the medical literature. In preliminary studies using mice with targeted deletion of AT1R genes that were generated in our laboratory, we became concerned about the specificity of these antibodies. Accordingly, we carried out a systematic evaluation of a panel of anti-AT1R antibodies that were purchased from commercial vendors, focusing on their utility and specificity for Western blot analysis and immunohistochemistry.

Materials and Methods

Please see online-only Data Supplement.

Results

We first performed Western blot analysis using homogenates of cortex (C) and medulla (M) from kidneys of wild-type (WT) mice comparing band patterns produced by 3 anti-AT1R antibodies. As shown in Figure 1, antibodies 1 and 2 each generated a single band between 38 and 48 kDa, which would be around the expected 41-kDa size of the AT1R.911 Although each of these antibodies identified a single band, the molecular weight of these bands was slightly different. In contrast, antibody 3 produced multiple bands of a broad range of sizes (Figure 1; n=3). Between the 3 antibodies, the patterns of reactivity were very different with no common bands seen within the predicted molecular size range for the AT1R.

Figure 1.

Figure 1. Western blot analysis of 40 μg homogenates of wild-type kidney cortex (C) and medulla (M) using 3 different commercially available angiotensin type 1 receptor (AT1R) antibodies. Antibody 1: Alomone AAR-011; antibody 2: Santa Cruz sc-1173 (n-10); antibody 3: Abcam 18801. X-ray films were exposed to the membranes for 2 min. Representative of n=2.

To test the specificity of each antibody for the AT1R, we performed additional Western blot analyses but now using protein homogenates from kidneys of mice genetically deficient in 1 or both AT1R subtypes. As shown in Figure 2, there were no apparent differences in the pattern of reactivity of each of the antibodies between protein extracts of kidneys from WT mice, compared with those from mice lacking the major AT1R isoform, AT1AR (AT1AKO). Furthermore, the patterns of antibody reactivity were virtually identical in the kidneys from WT and AT1abKO (Figure 2; n=3). To be certain that we were not missing a band corresponding to AT1R protein that was obscured or low abundance, we overexposed the X-ray film to the membrane for up to 60 minutes, but did not detect additional bands.

Figure 2.

Figure 2. Western blot analysis of kidney cortex and medulla from wild-type mice (WT), AT1AKO mice (A), and AT1ABKO mice (AB) using 3 different commercially available anti-AT1R antibodies. A, Antibody 1: Alomone AAR-011; B, antibody 2: Santa Cruz sc-1173 (n-10); C, antibody 3: AbCam 18801. Representative of n=3.

We also tested the polyclonal AT1R antibody 2 for immunohistochemical staining. As shown in Figure 3A3B, tissue sections from WT kidney in the absence (Figure 3A) of primary antibody demonstrate minimal background staining compared with sections incubated with the AT1R antibody (Figure 3B). Smooth muscle cells of the renal and arcuate arteries, interlobular (Figure 3B and 3F) and afferent arterioles (Figure 3C, 3E–3G), liver (Figure 3H), and adrenal gland (data not shown) were stained positively with the AT1R antibody on WT and AT1ABKO tissues. Prominent anti-AT1R immunostaining was also visualized in the proximal tubule brush border and basolateral membranes (Figure 3B–3E and 3G) of WT and AT1ABKO. Distal tubules, cortical, and medullary collecting ducts (Figure 3B–3E and 3G) also exhibited immunoreactivity. Thus, similar to the Western blotting data, patterns and localization of immunostaining with the AT1R antibody were identical in the kidneys of WT mice (Figure 3B–3D) and the AT1ABKO completely lacking AT1R (Figure 3E–3G).

Figure 3.

Figure 3. AT1R immunohistochemical localization using antibody 2 Santa Cruz N-10 rabbit polyclonal antibody (1:300) in kidney and liver of wild-type (WT) (A–D) mice and mice with combined deficiency of AT1A and AT1B receptors (AT1ABKO; E–H). Consecutive tissue sections from WT kidney in the absence (A) of primary antibody demonstrate minimal background immunostaining compared with incubation of the tissue section in the presence of the AT1R (B). AT1R was localized to vascular smooth muscle cells (arrow) of WT and AT1ABKO mice in renal cortical (A–G) and liver (H) tissues. Proximal tubule brush border and basolateral membrane (double arrowhead) and distal nephron segments (asterisk) demonstrated positive AT1R immunostaining in WT and AT1ABKO mice. Images were obtained using a ×100 oil immersion lens. Bar, 50 μm.

To verify that the AT1R knockout mice used in the studies were truly deficient in receptors, we measured AT1R expression and functional responses to angiotensin II. By quantitative real-time–polymerase chain reaction, AT1A mRNA receptor abundance in WT kidneys was similar in the cortex and medulla (10.7±0.4 and 12.5±1.5 arbitrary units for cortex and medulla, n=4; n.s.). In contrast, AT1A receptor transcript was undetectable in either cortex or medulla of both AT1AKO and AT1ABKO kidneys (Figure 4A). Additionally, AT1B receptor mRNA was detected in adrenal glands from WTs and AT1AKO but undetectable in AT1ABKO (Figure 4B). Furthermore, to confirm physiological absence of functional AT1R in the knockouts, we tested the ability of angiotensin II to cause acute increases in blood pressure. Bolus infusions of 10 μg/kg angiotensin II increased blood pressure by 34±4 mm Hg in WT mice. However, this response was significantly blunted in AT1AKO and completely absent in AT1ABKO mice (change: 4±1 and 0.5±0.8 mm Hg for AT1AKO and AT1ABKO; P<0.001 versus WTs, n=3–4; Figure 4C). These experiments confirm the lack of each AT1R subtype expression and activity in our knockout mouse lines and indicate that the anomalous results obtained by Western blotting and immunohistochemistry are likely a result of antibodies cross-reacting with unknown proteins other than the AT1R.

Figure 4.

Figure 4. A, CT values for AT1A receptor relative to GAPDH obtained by real-time polymerase chain reaction in kidney tissues from wild-type (WT), AT1AKO, and AT1ABKO mice. B, Representative agarose gel to visualize AT1B receptor mRNA in WT, AT1AKO, and AT1ABKO. C, Effect of acute angiotensin II infusion (10 μg/kg) on blood pressure in WT, AT1AKO, and AT1ABKO mice. P<0.005 vs WT; n=3 to 4.

To explore the possibility that the lack of specificity of anti-AT1R antibodies was attributable to insufficient sensitivity, we overexpressed the AT1R in human embryonic kidney (HEK) cells by transfecting them with DNA encoding the mouse AT1AR. To verify appropriate targeting of the AT1R protein to the plasma membrane, we used a plasmid encoding the AT1A receptor fused with the mCherry fluorescence protein (AT1A–mCherry) and imaged the receptor by live cell–fluorescence confocal microscopy. We found that, after 24 hours of transfection, a significant amount of the total AT1AR pool was present at the plasma membrane (Figure 5A, I). In addition, we also observed that the nonplasma membrane–associated AT1AR pool does not colocalize with the endoplasmic reticulum–associated protein Calreticulin (Figure 5A, II and III), but it partially colocalizes with the trans-Golgi network-associated protein GalNac-T (Figure 5A, IV–VI).

Figure 5.

Figure 5. A, Cellular localization of AT1A in HEK cells cotransfected with expression plasmids encoding AT1A receptor fused with fluorescent mCherry (AT1A, magenta, I, III, IV, and VI) and with different plasmids encoding fluorescent markers for endoplasmic reticulum (Calreticulin, green, II and III) or Golgi apparatus (GalNAc, green, V and VI). The fluorescent constructs were visualized after 24 h by live-cell confocal microscopy. AT1A receptors localize in the plasma membrane and partially associate with the Golgi apparatus. The results are representative of ≥2 independent experiments. B, Detection of AT1 receptors in HEK cells transfected with varying amounts (0, 2, 4, and 8 μg) of a plasmid encoding the His-tagged AT1A receptor (AT1A-His DNA) using different antibodies. Anti-His penta His antibody, Qiagen 34660; anti-AT1 receptor 1: Alomone AAR-011; anti-AT1 receptor 2: Santa Cruz sc-1173 (n-10); anti-AT1 receptor 3: AbCam 18801. Red boxes indicate bands corresponding to the AT1AR protein. Asterisk indicates a band recognized by the His antibody in nontransfected cells, suggesting an endogenous non-specific band.

We next tested the ability of the commercial antibodies to detect AT1AR increments at different levels of over-expression. For this, we subcloned the AT1AR sequence from plasmid AT1A-mCherry into the pcDNA3.1 His vector in frame with the His epitope (AT1A-His), and different amounts of plasmidic DNA were transfected into HEK cells. For detection of the exogenous His-tagged proteins by Western blot, an anti-His antibody was used. The anti-His antibody detected multiple bands close to 39 kDa in cells transfected with 2 μg plasmid. The intensity of these bands (relative to GAPDH) increased by 83±40% and 193±39% (n=3) in cells transfected with 4 and 8 μg DNA (lower red box on Figure 5B). In contrast, this pattern was not reproduced by utilizing any of the anti-AT1R antibodies (Figure 5B).

Discussion

In this study, we tested 3 different rabbit polyclonal commercial antibodies that have been used in published reports to detect AT1R protein. Antibodies 1 and 2 were raised against a short sequence (15 amino acids) of the extracellular amino terminus of the AT1R protein that is identical among rat and mouse. This region is ≈95% identical between AT1A and AT1B receptors (accession numbers NP_796296.1 and EDL34899.1). Antibody 3 was raised against intracellular carboxy terminus sequences also identical between rat and mouse. In this case, 2 synthetic peptides specific for each receptor subtype, AT1A and AT1B, were used. The AT1A and AT1B receptors are each 359 amino acids, highly homologous (sharing <94% amino acid identity) with predicted molecular masses of 41 kDa. Therefore, the antibodies used in these studies should not distinguish AT1A and AT1B receptor subtypes. Although both AT1R isoforms undergo post-translational glycosylation, a single ≈41 kDa band should represent the nonglycosylated AT1A or AT1B receptor, whereas higher molecular bands would be expected for the glycosylated forms.12

By performing direct side-by-side comparisons of the bands recognized by each antibody, we concluded that each antibody binds to distinct unknown proteins of diverse molecular sizes and raised the concern that these antibodies cross-react with proteins other than the AT1R. Experiments using kidney tissue from mice with genetic deficiencies of the major murine AT1R isoform (AT1A) revealed that all 3 antibodies showed the same pattern of bands on Western blots whether the proteins were derived from WT or AT1AKO mice. Similar findings using the single AT1AKO were recently reported.12a Inclusion of samples from the double knockout (AT1ABKO) eliminated the possibility that the positive signal in the AT1AKO was attributable to upregulation of the homologous AT1BR. This suggests that none of these antibodies recognize the AT1R protein with sufficient specificity in kidney tissue by Western blot.

Our immunohistochemical staining studies revealed apparent AT1R localization in the renal vasculature and proximal and distal tubules. However, identical staining patterns were observed when kidneys from mice lacking both AT1R subtypes (AT1ABKO) were used. The positive AT1R immunostaining in the renal microvasculature of the AT1ABKO kidneys was unexpected because these tissues lack renal vasoconstrictor responses to angiotensin II in in vitro and in vivo.13,14 Additionally, the apparent positive AT1R immunostaining was also observed in the liver vasculature of AT1ABKO mice, indicating that the nonspecific positive antibody staining observed in the vascular smooth muscle of the AT1ABKO is not restricted to the kidney. The identity of the proteins recognized by these antibodies remains elusive. We conducted immunoprecipitation of kidney homogenates (using antibodies 1 and 2) followed by mass spectrometry and were unable to identify either the AT1R or any other protein in the 41-kDa range. Two possibilities may explain these inconclusive results: (1) the antibodies are not suitable for immunoprecipitation experiments; and (2) the nonspecific protein has yet to be reported and, therefore, does not exist in the current peptide database. Nonetheless, despite our inability to identify the specific protein or proteins identified by these antibodies, the cumulative results from our studies provide compelling evidence that this protein is not the AT1R.

We were confident that these findings were intrinsic to the antibodies and not to the presence of AT1R in our knockout lines because we verified the absence of AT1R mRNA expression and functional responses to angiotensin II in randomly selected mice from our colony. Based on the strategy of gene disruption used to generate our KO mice14,15 where many stop codons were introduced into the early portions of the coding sequence of each gene, it is theoretically possible that very small, truncated forms of the receptors might be generated. However, it is highly unlikely that these protein fragments would reach the cell surface. Moreover, if present, they should appear as unique bands in the knockout mice with sizes substantially smaller than that predicted for the native AT1R protein.14,15

We used HEK cells over-expressing AT1A receptors to test the antibodies again. Our confocal fluorescence images in living HEK cells show proper plasma membrane expression of the AT1A receptor. In addition, AT1A–mCherry fluorescence was not significant in the endoplasmic reticulum, indicating that processed AT1AR proteins were properly sorted to the Golgi apparatus for further secretion, as shown by colocalization with the marker for this organelle. These findings indicate that HEK cells are able to transcribe, translate, and normally sort the AT1AR, which localizes normally to the plasma membrane. Accordingly, we used HEK cells transfected with the AT1AR–His plasmid. Using an anti-His antibody, we detected distinct amounts of His-tagged AT1AR proportional to the amount of DNA transfected under each experimental condition. Bands of molecular weights in the ≈39-kDa and ≈65-kDa range were detected only in cells transfected with the AT1A-His plasmid (red boxes on Figure 5B). A single band at ≈51 kDa was present in all samples, including the mock-transfected cells, and likely represents endogenous His proteins expressed by HEK cells (marked with an asterisk on Figure 5B). Nevertheless, the ≈39-kDa band is consistent with the nonglycosylated form of the AT1R12 even though one may question the apparent molecular weight of AT1R “monomer” because this band appears to be slightly smaller than the predicted 41 kDa mass of this protein. This small apparent difference in size could be accounted for by an altered migration velocity due to the positively charged histidines added to the carboxyl-terminal tail. Furthermore, the multiple bands apparent at ≈39 kDa could also be proteolytic degradation products of the AT1R protein. Additionally, although the multiple banding at ≈65 kDa is consistent with the different degrees of AT1R glycosylation reported for the rat kidney,16 it is also possible that these bands represent ubiquitinated receptor which is the mechanism whereby the AT1R is degraded. Nevertheless, in contrast to the anti-His antibody, these bands were not identified by any of the anti-AT1R antibodies. Although we do not observe specific reactivity of the antibodies to AT1R protein expressed at high levels in HEK cells, the failure of the antibodies to detect the AT1R in Western blots does not appear to be an issue of insufficient sensitivity. It is important to note that the nonspecific bands recognized by the anti-AT1R antibodies in HEK cells are different in number and size compared with those observed in the kidney tissues (Figure 2), which provides additional evidence for the lack of specificity of these antibodies. Although the inability of the commercial antibodies to recognize the AT1R protein could be explained by potential masking of the epitope by the His tag, we believe this is unlikely to occur because of the denaturing conditions used to unfold the proteins in our Western blotting technique. Additionally, the His tag is attached at the carboxy terminus region of the protein, and at least 2 of the antibodies tested are raised against the amino terminus sequence.

Other investigators attempted to generate custom-made antibodies without success. The Daugherty laboratory8 generated several antisera against potential new antigenic sites within the AT1R receptor, and they were not able to demonstrate any specific interaction to the AT1A or AT1B receptors by either Western blotting or immunostaining of tissue sections. In a different report by Hoffmann et al,17 investigators generated antibodies using the last 20 amino acids of the carboxy terminus tail of each AT1A and AT1B receptors. These antibodies yielded bands of different molecular mass in neural cells compared with mammalian tissues. Although these bands were absent when preincubating the antibodies with the immunizing synthetic peptides, this strategy alone is insufficient to demonstrate specificity for the intact receptor protein, and thus the specificity of such antibody remains elusive. Finally, using a monoclonal antibody (6313/G2) raised against and N-terminal peptide (residues 8–17), we previously localized the AT1R protein in renal vascular smooth muscle cells, proximal tubule, and more distal nephron segments in kidneys of adult rats.16,18 The specificity of this antibody was confirmed by Western blotting of COS-7 cells transfected with an AT1R-expressing plasmid.19 Thus, it is possible that identification of the AT1R by antibody-based techniques is suitable for rat but not mouse tissues. In contrast, the Bernstein laboratory raised a polyclonal antibody against N-terminal peptide residues 15–24, which stained mainly vascular smooth muscle cells, brush border, and thick ascending limbs.20 Unlike our report, this antibody failed to demonstrate immunoreactivity in distal collecting ducts. The origin of this discrepancy is still unknown, but it may be explained by some degree of antibody cross-reactivity. Unfortunately, the identity of the peptide being recognized in such studies has not been verified likely because AT1KO mice were not available at the time.

Perspectives

We report here that commercially available antibodies are erroneous tools to detect the AT1R protein. Generation of highly specific antibodies for G protein–coupled receptors has reportedly been difficult,2126 and the reasons behind this are yet to be understood. One explanation is potential differences in structure and charge between the glycosylation (naturally carried in the AT1R) and the synthetic peptides used when raising these antibodies. Successful strategies to generate reliable anti-AT1R antibodies are urgently needed. Investigators should use alternative methods such as ligand-binding,1 epitope-tagging,27 Northern blot,28 or quantitative real-time–polymerase chain reaction when studying the biology of AT1R. Interpretations of previously published work relying solely on quantitative and qualitative assessments of the AT1R protein using these antibodies should be viewed with extreme caution.

Footnotes

The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.112.203679/-/DC1.

Correspondence to Marcela Herrera, Division of Nephrology, Duke University Medical Center, Box 103015, Durham, NC 27710. E-mail

References

  • 1. Gurley SB, Riquier-Brison AD, Schnermann J, Sparks MA, Allen AM, Haase VH, Snouwaert JN, Le TH, McDonough AA, Koller BH, Coffman TM. AT1A angiotensin receptors in the renal proximal tubule regulate blood pressure.Cell Metab. 2011; 13:469–475.CrossrefMedlineGoogle Scholar
  • 2. Crowley SD, Gurley SB, Herrera MJ, Ruiz P, Griffiths R, Kumar AP, Kim HS, Smithies O, Le TH, Coffman TM. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney.Proc Natl Acad Sci USA. 2006; 103:17985–17990.CrossrefMedlineGoogle Scholar
  • 3. Crowley SD, Zhang J, Herrera M, Griffiths R, Ruiz P, Coffman TM. Role of AT1 receptor-mediated salt retention in angiotensin II-dependent hypertension.Am J Physiol Renal Physiol. 2011; 301:F1124–F1130.CrossrefMedlineGoogle Scholar
  • 4. Lai EY, Solis G, Luo Z, Carlstrom M, Sandberg K, Holland S, Wellstein A, Welch WJ, Wilcox CS. p47(phox) is required for afferent arteriolar contractile responses to angiotensin II and perfusion pressure in mice.Hypertension. 2012; 59:415–420.LinkGoogle Scholar
  • 5. Masuda T, Muto S, Fujisawa G, Iwazu Y, Kimura M, Kobayashi T, Nonaka-Sarukawa M, Sasaki N, Watanabe Y, Shinohara M, Murakami T, Shimada K, Kobayashi E, Kusano E. Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy.Am J Physiol Heart Circ Physiol. 2012; 302:H1871–H1883.CrossrefMedlineGoogle Scholar
  • 6. Polichnowski AJ, Lu L, Cowley AW. Renal injury in angiotensin II+L-NAME-induced hypertensive rats is independent of elevated blood pressure.Am J Physiol Renal Physiol. 2011; 300:F1008–F1016.CrossrefMedlineGoogle Scholar
  • 7. Varagic J, Frohlich ED, Susic D, Ahn J, Matavelli L, López B, Díez J. AT1 receptor antagonism attenuates target organ effects of salt excess in SHRs without affecting pressure.Am J Physiol Heart Circ Physiol. 2008; 294:H853–H858.CrossrefMedlineGoogle Scholar
  • 8. Rateri DL, Moorleghen JJ, Balakrishnan A, Owens AP, Howatt DA, Subramanian V, Poduri A, Charnigo R, Cassis LA, Daugherty A. Endothelial cell-specific deficiency of Ang II type 1a receptors attenuates Ang II-induced ascending aortic aneurysms in LDL receptor−/− mice.Circ Res. 2011; 108:574–581.LinkGoogle Scholar
  • 9. Iwai N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y, Inagami T. Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression.Biochem Biophys Res Commun. 1991; 177:299–304.CrossrefMedlineGoogle Scholar
  • 10. Kakar SS, Sellers JC, Devor DC, Musgrove LC, Neill JD. Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation.Biochem Biophys Res Commun. 1992; 183:1090–1096.CrossrefMedlineGoogle Scholar
  • 11. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor.Nature. 1991; 351:233–236.CrossrefMedlineGoogle Scholar
  • 12. Deslauriers B, Ponce C, Lombard C, Larguier R, Bonnafous JC, Marie J. N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane.Biochem J. 1999; 339(pt 2):397–405.CrossrefMedlineGoogle Scholar
  • 12a. Benicky J, Hafko R, Sanchez-Lemus E, Aguilera G, Saavedra JM. Six Commercially Available Angiotensin II AT(1) Receptor Antibodies are Non-specific.Cell Mol Neurobiol. July 28, 2012. DOI: 10.1007/s10571-012-9862-y. http://www.springerlink.com/content/c124121217554717/. Accessed November 2, 2012.CrossrefMedlineGoogle Scholar
  • 13. Park S, Bivona BJ, Harrison-Bernard LM. Compromised renal microvascular reactivity of angiotensin type 1 double null mice.Am J Physiol Renal Physiol. 2007; 293:F60–F67.CrossrefMedlineGoogle Scholar
  • 14. Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman TM. Regulation of blood pressure by the type 1A angiotensin II receptor gene.Proc Natl Acad Sci USA. 1995; 92:3521–3525.CrossrefMedlineGoogle Scholar
  • 15. Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, Coffman TM. Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci USA. 1998; 95:15496–15501.CrossrefMedlineGoogle Scholar
  • 16. Harrison-Bernard LM, Imig JD, Carmines PK. Renal AT1 receptor protein expression during the early stage of diabetes mellitus.Int J Exp Diabetes Res. 2002; 3:97–108.CrossrefMedlineGoogle Scholar
  • 17. Hoffmann A, Cool DR. Characterization of two polyclonal peptide antibodies that recognize the carboxy terminus of angiotensin II AT1A and AT1B receptors.Clin Exp Pharmacol Physiol. 2005; 32:936–943.CrossrefMedlineGoogle Scholar
  • 18. Harrison-Bernard LM, Navar LG, Ho MM, Vinson GP, el-Dahr SS. Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody.Am J Physiol. 1997; 273(1 pt 2):F170–F177.MedlineGoogle Scholar
  • 19. Barker S, Marchant W, Ho MM, Puddefoot JR, Hinson JP, Clark AJ, Vinson GP. A monoclonal antibody to a conserved sequence in the extracellular domain recognizes the angiotensin II AT1 receptor in mammalian target tissues.J Mol Endocrinol. 1993; 11:241–245.CrossrefMedlineGoogle Scholar
  • 20. Paxton WG, Runge M, Horaist C, Cohen C, Alexander RW, Bernstein KE. Immunohistochemical localization of rat angiotensin II AT1 receptor.Am J Physiol. 1993; 264(6 pt 2):F989–F995.MedlineGoogle Scholar
  • 21. Pradidarcheep W, Labruyère WT, Dabhoiwala NF, Lamers WH. Lack of specificity of commercially available antisera: better specifications needed.J Histochem Cytochem. 2008; 56:1099–1111.CrossrefMedlineGoogle Scholar
  • 22. Rhodes KJ, Trimmer JS. Antibodies as valuable neuroscience research tools versus reagents of mass distraction.J Neurosci. 2006; 26:8017–8020.CrossrefMedlineGoogle Scholar
  • 23. Lu X, Bartfai T. Analyzing the validity of GalR1 and GalR2 antibodies using knockout mice.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379:417–420.CrossrefMedlineGoogle Scholar
  • 24. Jensen BC, Swigart PM, Simpson PC. Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379:409–412.CrossrefMedlineGoogle Scholar
  • 25. Hamdani N, van der Velden J. Lack of specificity of antibodies directed against human beta-adrenergic receptors.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379:403–407.CrossrefMedlineGoogle Scholar
  • 26. Pradidarcheep W, Stallen J, Labruyère WT, Dabhoiwala NF, Michel MC, Lamers WH. Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors.Naunyn Schmiedebergs Arch Pharmacol. 2009; 379:397–402.CrossrefMedlineGoogle Scholar
  • 27. Roy SF, Laporte SA, Escher E, Leduc R, Guillemette G. Epitope tagging and immunoreactivity of the human angiotensin II type 1 receptor.Can J Physiol Pharmacol. 1997; 75:690–695.CrossrefMedlineGoogle Scholar
  • 28. Burson JM, Aguilera G, Gross KW, Sigmund CD. Differential expression of angiotensin receptor 1A and 1B in mouse.Am J Physiol. 1994; 267(2 pt 1):E260–E267.MedlineGoogle Scholar

Novelty and Significance

What Is New?

  • Antibody-based detection of AT1R is unreliable as a result of the lack of specificity of commercial antibodies that recognize proteins different than the AT1R.

What Is Relevant?

  • The AT1R mediates the hypertensive actions of angiotensin II. Understanding AT1R protein tissue distribution, abundance, and interaction with other molecules is crucial to elucidate the pathophysiology of cardiovascular and renal diseases and develop new pharmacologic tools for their treatment.

  • Many investigators have published studies using these unreliable antibodies.

  • Caution must be used when interpreting such reports and designing experimental approaches to understand the biology of these receptors.

Summary

Our studies indicate that commercially available antibodies may not always be suitable for detecting AT1R protein. Inclusion of appropriate positive and negative controls is essential when using antibody-based techniques. Investigators should be wary of using these tools when designing new experimental approaches to understand the biology and pathophysiology of AT1 receptors.

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