Six Commercially Available Angiotensin II AT1 Receptor Antibodies are Non-specific

被引:92
作者
Benicky, Julius [1 ]
Hafko, Roman [1 ]
Sanchez-Lemus, Enrique [1 ]
Aguilera, Greti [2 ]
Saavedra, Juan M. [1 ]
机构
[1] NIMH, Pharmacol Sect, DIRP, NIH, Bethesda, MD 20892 USA
[2] NICHD, Sect Endocrine Physiol, PDEGEN, NIH,CRC, Bethesda, MD 20892 USA
关键词
GPCR receptor antibodies; Antibody characterization; Angiotensin II receptors; Renin-angiotensin System; AT(1) receptor immunocytochemistry; AT(1) receptor antibodies; AT(1A) receptor knock-out; BLOOD-PRESSURE REGULATION; HYPOTHALAMIC CELL-LINE; ADRENERGIC-RECEPTORS; DEFICIENT MICE; RAT-BRAIN; EXPRESSION; SPECIFICITY; SUBTYPES; MOUSE; GENE;
D O I
10.1007/s10571-012-9862-y
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Commercially available Angiotensin II AT(1) receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT(1) receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT(1) receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT(1) receptor. However, identical bands were observed in wild-type mice and in AT(1A) knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT(1) receptors or transfected with AT(1A) receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT(1A) knock-out and wild-type mice and in 4B cells with or without AT(1) receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT(1) receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT(1) receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT(1) receptor physiology in the absence of full antibody characterization.
引用
收藏
页码:1353 / 1365
页数:13
相关论文
共 39 条
[1]   Tissue Renin-Angiotensin-Aldosterone Systems: Targets for Pharmacological Therapy [J].
Bader, Michael .
ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, 2010, 50 :439-465
[2]   Should we be cautious on the use of commercially available antibodies to dopamine receptors? [J].
Bodei, Serena ;
Arrighi, Nicola ;
Spano, PierFranco ;
Sigala, Sandra .
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 2009, 379 (04) :413-415
[3]   DIFFERENTIAL EXPRESSION OF ANGIOTENSIN RECEPTOR 1A AND 1B IN MOUSE [J].
BURSON, JM ;
AGUILERA, G ;
GROSS, KW ;
SIGMUND, CD .
AMERICAN JOURNAL OF PHYSIOLOGY, 1994, 267 (02) :E260-E267
[4]   THE LIGAND-BINDING SIGNATURES OF THE RAT AT(1A), AT(1B) AND THE HUMAN AT(1) RECEPTORS ARE ESSENTIALLY IDENTICAL [J].
CHIU, AT ;
DUNSCOMB, J ;
KOSIEROWSKI, J ;
BURTON, CRA ;
SANTOMENNA, LD ;
CORJAY, MH ;
BENFIELD, P .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1993, 197 (02) :440-449
[5]   TISSUE-SPECIFIC EXPRESSION OF TYPE-1 ANGIOTENSIN-II RECEPTOR SUBTYPES - AN IN-SITU HYBRIDIZATION STUDY [J].
GASC, JM ;
SHANMUGAM, S ;
SIBONY, M ;
CORVOL, P .
HYPERTENSION, 1994, 24 (05) :531-537
[6]   Lack of specificity of antibodies directed against human beta-adrenergic receptors [J].
Hamdani, Nazha ;
van der Velden, Jolanda .
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 2009, 379 (04) :403-407
[7]   Characterization and distribution of angiotensin II receptor subtypes in the mouse brain [J].
Häuser, W ;
Jöhren, O ;
Saavedra, JM .
EUROPEAN JOURNAL OF PHARMACOLOGY, 1998, 348 (01) :101-114
[8]  
INAGAMI T, 1994, J HYPERTENS, V12, pS83
[9]   REGULATION OF BLOOD-PRESSURE BY THE TYPE 1A ANGIOTENSIN-II RECEPTOR GENE [J].
ITO, M ;
OLIVERIO, MI ;
MANNON, PJ ;
BEST, CF ;
MAEDA, N ;
SMITHIES, O ;
COFFMAN, TM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (08) :3521-3525
[10]   Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific [J].
Jensen, Brian C. ;
Swigart, Philip M. ;
Simpson, Paul C. .
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 2009, 379 (04) :409-412