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

Abstract

HomeHypertensionVol. 61, No. 4Lack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type-1 Receptor Protein Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBLack of Specificity of Commercial Antibodies Leads to Misidentification of Angiotensin Type-1 Receptor Protein Katherine J. Elliott, Keita Kimura and Satoru Eguchi Katherine J. ElliottKatherine J. Elliott Cardiovascular Research CenterTemple University School of MedicinePhiladelphia, Pennsylvania Search for more papers by this author , Keita KimuraKeita Kimura Cardiovascular Research CenterTemple University School of MedicinePhiladelphia, Pennsylvania Search for more papers by this author and Satoru EguchiSatoru Eguchi Cardiovascular Research CenterTemple University School of MedicinePhiladelphia, Pennsylvania Search for more papers by this author Originally published4 Feb 2013https://doi.org/10.1161/HYPERTENSIONAHA.111.00943Hypertension. 2013;61:e31Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2013: Previous Version 1 To the Editor:We agree with the report by Herrera et al1 demonstrating that antiangiotensin II type-1 receptor (AT1R) antibodies that are commonly used exhibit nonspecific binding that may lead to erroneous results. Because only 3 AT1R antibodies were used in the study, we would like to share our findings to support their conclusion.Antihemagglutinin antibody detected a major band (≈40 kDa) in human embryonic kidney cells expressing rat AT1AR fused with hemagglutinin tag at either amino terminus or carboxyl terminus but not in the control cells. The molecular weight is consistent with the AT1AR band observed by Herrera et al.1 In contrast, no specific difference was observed among the cell lysates immunoblotted with 3 distinct AT1R antibodies, which should have detected rat AT1AR according to the manufacturers (Figure).Herrera et al1 used mouse tissues and cells expressing mouse AT1AR for their experiments. Our data support and expand the findings by including rat AT1AR and adding 2 other frequently used AT1R antibodies. Researchers must be aware that commercially available AT1R antibodies and antibodies against other G protein–coupled receptors may not be specific enough to quantify the receptor expression. To publish an article using an AT1R antibody, it should be accompanied by sufficient evidence to support the specificity. We also encourage the antibody manufacturers to include rigorous confirmation before commercialization.Katherine J. ElliottKeita KimuraSatoru EguchiCardiovascular Research CenterTemple University School of MedicinePhiladelphia, PennsylvaniaSources of FundingThis work was supported by National Institutes of Health grant HL076770.DisclosuresNone.Download figureDownload PowerPointFigure. Detection of angiotensin II type 1A receptor (AT1AR) expressed in human embryonic kidney (HEK) cells. A, HEK cells were transfected with an expression vector encoding rat AT1AR fused with hemagglutinin (HA) tag at amino terminus or control green fluorescent protein (GFP) for 48 hours. The cells were serum-starved for the last 24 hours and stimulated with 100 nmol/L angiotensin II for 10 minutes. Phospho-extracellular signal-regulated kinase (ERK-p) antibody (Santa Cruz 7383) blotting demonstrated functional expression of AT1AR. Immunoblotting was also performed with Abcam antibody (ab9391) raised against carboxyl-terminal sequences of AT1R or Invitrogen antibody (71–5500) against HA tag. Representative blots are shown from 2 independent experiments. B, HEK cells were transfected with an expression vector encoding rat AT1AR fused with HA tag at carboxyl terminus or control red fluorescent protein (RFP) for 48 hours. Immunoblotting was performed with HA antibody, Abcam AT1R antibody (ab9391), Santa Cruz antibody (1173) raised against amino-terminal sequences of AT1R, or another antibody from Santa Cruz (579) raised against carboxyl-terminal sequences of AT1R.FootnotesLetters to the Editor will be published, if suitable, as space permits. They should not exceed 500 words (typed double-spaced) plus 5 references in length and may be subject to editing or abridgment.