title: Monoclonal antibodies to discriminate the EF hand containing calcium binding adaptor proteins EFhd1 and EFhd2.
abstract: Small Ca(2+) binding adaptor proteins of the EF hand family play important roles in neuronal and immune cell Ca(2+) signaling. Swiprosin-1/EFhd2 (EFhd2) and Swiprosin-2/EFhd1 (EFhd1) are conserved and very homologous Ca(2+) binding adaptor proteins of the EF hand family, with possibly redundant functions. In particular, EFhd2 has been proposed to be involved in B cell signaling and neuropathological disorders. Little is known thus far about the expression of EFhd2 on the single cell level in tissue sections or blood cells. Here we describe the generation of four specific anti-EFhd2 monoclonal antibodies. These recognize murine and human EFhd2, but not murine EFhd1, and their binding site maps to a region in the N-terminal part of EFhd2, where EFhd2 and EFhd1 differ most. Moreover, to detect EFhd1 specifically, we also generated anti-EFhd1 polyclonal antibodies, making use of a singular peptide of the N-terminal part of the protein. Using anti-EFhd2 MAb, we reveal two EFhd2 pools in B cells, one at the membrane and one cytoplasmic pool. Staining of human peripheral blood mononuclear cells shows EFhd2 expression in B cells but a ∼5 fold higher expression in monocytes. Taken together, EFhd2 monoclonal antibodies will be valuable to assess the real subcellular localization and expression level of EFhd2 in healthy and diseased primary cells and tissues.
authors.affiliation: Division of Molecular Immunology, Department of Medicine III, University of Erlangen-Nürnberg, Nikolaus Fiebiger Center, Erlangen, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brachs
authors.forename: Christiane
authors.initials: C
authors.lastname: Lang
authors.forename: Rolf
authors.initials: R
authors.lastname: Buslei
authors.forename: Pavitra
authors.initials: P
authors.lastname: Purohit
authors.forename: Barbara
authors.initials: B
authors.lastname: Fürnrohr
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Jäck
authors.forename: Dirk
authors.initials: D
authors.lastname: Mielenz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; C489648:EFHC1 protein, human; C502299:EFHD2 protein, human; D011994:Recombinant Proteins
references.citation: Microbes Infect. 2010 Aug;12(8-9):683-9
references.pmid: 20417300
references.citation: Int Immunol. 1992 Aug;4(8):831-40
references.pmid: 1419955
references.citation: J Immunol. 2005 Mar 15;174(6):3508-17
references.pmid: 15749887
references.citation: Eur Arch Psychiatry Clin Neurosci. 2009 Apr;259(3):151-63
references.pmid: 19165527
references.citation: Cell Death Differ. 2007 Nov;14(11):1936-47
references.pmid: 17673920
references.citation: Cell Calcium. 2012 May;51(5):411-7
references.pmid: 22317918
references.citation: J Proteome Res. 2007 Sep;6(9):3752-9
references.pmid: 17705417
references.citation: J Immunol. 2010 Apr 1;184(7):3665-76
references.pmid: 20194721
references.citation: Curr Alzheimer Res. 2010 Sep;7(6):549-55
references.pmid: 20455864
references.citation: Cell. 1981 Dec;27(2 Pt 1):381-90
references.pmid: 6277505
references.citation: J Immunol. 1981 Jun;126(6):2466-9
references.pmid: 6785356
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Immunol Res. 1986;5(4):263-70
references.pmid: 3110331
references.citation: J Cell Sci. 2011 Oct 1;124(Pt 19):3266-78
references.pmid: 21896648
references.citation: FASEB J. 2011 Nov;25(11):4011-23
references.pmid: 21795503
references.citation: J Neurochem. 2008 Jul;106(1):96-106
references.pmid: 18346207
references.citation: Cell Tissue Kinet. 1979 Jan;12(1):71-80
references.pmid: 84712
references.citation: Cell Commun Signal. 2011 Jan 18;9:2
references.pmid: 21244694
references.citation: Protein Pept Lett. 2013 May;20(5):573-83
references.pmid: 22973849
references.citation: Acta Neuropathol. 2007 May;113(5):585-90
references.pmid: 17221204
references.citation: Mol Cell Biol. 1985 Dec;5(12):3610-6
references.pmid: 3915782
references.citation: Biochim Biophys Acta. 2004 Jan 14;1696(1):121-9
references.pmid: 14726212
references.citation: Adv Exp Med Biol. 2010;685:9-20
references.pmid: 20687491
references.citation: J Neurochem. 2006 Jan;96(1):292-304
references.pmid: 16336229
references.citation: J Immunol. 2004 Aug 1;173(3):1858-67
references.pmid: 15265918
references.citation: J Immunol. 2003 Sep 15;171(6):3091-101
references.pmid: 12960335
references.citation: J Gene Med. 2006 Aug;8(8):1037-47
references.pmid: 16779863
references.citation: Biochem Soc Trans. 2011 Aug;39(4):868-73
references.pmid: 21787315
references.citation: Neurobiol Aging. 2011 Oct;32(10):1839-48
references.pmid: 20096956
references.citation: FEBS J. 2009 Jun;276(12):3308-23
references.pmid: 19438725