title: Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry.
abstract: Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.
authors.affiliation: Department of Chemistry and Molecular Biology and the Center for Protease Research, North Dakota State University, Fargo, ND 58108-6050, United States.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Hermann
authors.forename: Travis
authors.initials: T
authors.lastname: Van der Steen
authors.forename: Emilie E
authors.initials: EE
authors.lastname: Vomhof-Dekrey
authors.forename: Sejaa
authors.initials: S
authors.lastname: Al-Badrani
authors.forename: Steve B
authors.initials: SB
authors.lastname: Wanjara
authors.forename: Jarrett J
authors.initials: JJ
authors.lastname: Failing
authors.forename: Jodie S
authors.initials: JS
authors.lastname: Haring
authors.forename: Glenn P
authors.initials: GP
authors.lastname: Dorsam
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
chemical_list: D000906:Antibodies; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D012313:RNA
references.citation: Biochim Biophys Acta. 1997 Jun 27;1357(2):249-55
references.pmid: 9223629
references.citation: Blood. 2008 Feb 15;111(4):1885-93
references.pmid: 18000164
references.citation: J Immunol. 2000 Feb 1;164(3):1200-10
references.pmid: 10640731
references.citation: Ann N Y Acad Sci. 1998 Dec 11;865:397-407
references.pmid: 9928038
references.citation: J Immunol. 2001 Mar 1;166(5):2907-12
references.pmid: 11207237
references.citation: Symp Soc Exp Biol. 1979;33:9-36
references.pmid: 524275
references.citation: J Exp Med. 1984 Oct 1;160(4):1054-69
references.pmid: 6090573
references.citation: Immunology. 2002 May;106(1):87-95
references.pmid: 11972636
references.citation: J Pharmacol Exp Ther. 2002 Apr;301(1):37-50
references.pmid: 11907155
references.citation: Brain Behav Immun. 2008 Oct;22(7):1032-1040
references.pmid: 18534815
references.citation: J Immunol. 2001 Feb 15;166(4):2522-30
references.pmid: 11160313
references.citation: Reproduction. 2007 Aug;134(2):281-92
references.pmid: 17660238
references.citation: Biochem Soc Trans. 2002 Aug;30(4):441-6
references.pmid: 12196111
references.citation: Int Immunol. 2006 Dec;18(12):1691-700
references.pmid: 17077178
references.citation: Cancer Res. 2000 Jun 1;60(11):3105-12
references.pmid: 10850463
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: J Immunol. 1999 Feb 1;162(3):1707-16
references.pmid: 9973433
references.citation: J Neuroimmunol. 1996 Aug;68(1-2):27-38
references.pmid: 8784257
references.citation: Neuron. 1992 Apr;8(4):811-9
references.pmid: 1314625
references.citation: Mol Cell Neurosci. 1994 Apr;5(2):145-52
references.pmid: 7518311
references.citation: Cell Mol Life Sci. 2009 Oct;66(20):3337-52
references.pmid: 19629391
references.citation: Ann N Y Acad Sci. 1996 Dec 26;805:753-9
references.pmid: 8993473
references.citation: Eur J Pharmacol. 1996 Apr 29;302(1-3):207-14
references.pmid: 8791009
references.citation: FASEB J. 1991 May;5(8):2145-54
references.pmid: 1850705
references.citation: Gastroenterology. 2003 Apr;124(4):961-71
references.pmid: 12671893
references.citation: Mol Pharmacol. 2006 Jan;69(1):363-73
references.pmid: 16244179
references.citation: Neuroscience. 1979;4(12):1953-76
references.pmid: 394023
references.citation: Mol Endocrinol. 2008 Jan;22(1):147-55
references.pmid: 17885205
references.citation: Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2939-43
references.pmid: 7708752
references.citation: J Nucl Med. 2008 Jan;49(1):112-21
references.pmid: 18077536
references.citation: J Biol Chem. 2005 Jul 29;280(30):28034-43
references.pmid: 15932876
references.citation: Exp Hematol. 2002 Sep;30(9):1001-9
references.pmid: 12225791
references.citation: Peptides. 1997;18(10):1547-54
references.pmid: 9437715
references.citation: Brain Behav Immun. 1987 Jun;1(2):148-58
references.pmid: 3452442
references.citation: Regul Pept. 1997 Sep 26;72(1):41-54
references.pmid: 9404731
references.citation: Brain Behav Immun. 2008 Oct;22(7):1024-1031
references.pmid: 18555660
references.citation: J Immunol. 1985 Aug;135(2 Suppl):755s-765s
references.pmid: 2861231
references.citation: J Pharmacol Exp Ther. 2003 Aug;306(2):638-45
references.pmid: 12750439
references.citation: J Immunol. 2009 Oct 1;183(7):4346-59
references.pmid: 19734220
references.citation: Arthritis Rheum. 2008 Apr;58(4):1010-9
references.pmid: 18383379
references.citation: Arthritis Rheum. 1999 Nov;42(11):2418-29
references.pmid: 10555038
references.citation: Mol Immunol. 2010 Mar;47(6):1181-94
references.pmid: 20117839
references.citation: Regul Pept. 2009 Nov 27;158(1-3):68-76
references.pmid: 19729043
references.citation: J Clin Invest. 1974 Sep;54(3):536-44
references.pmid: 4369434
references.citation: J Neuroimmunol. 1993 Oct;48(1):59-69
references.pmid: 7901234
references.citation: J Immunol. 1983 Sep;131(3):1173-7
references.pmid: 6309960
references.citation: Biochem Soc Trans. 2007 Aug;35(Pt 4):724-8
references.pmid: 17635134
references.citation: J Immunol. 1999 Feb 15;162(4):2103-12
references.pmid: 9973484
references.citation: J Immunol. 1994 Aug 15;153(4):1762-8
references.pmid: 7519212
references.citation: Clin Cancer Res. 2004 Dec 15;10(24):8235-42
references.pmid: 15623599
references.citation: J Biol Chem. 1978 Dec 25;253(24):8667-70
references.pmid: 214427