title: The sodium-dependent inorganic phosphate transporter SLC34A1 (NaPi-IIa) is not localized in the mouse brain: a case of tissue-specific antigenic cross-reactivity.
abstract: The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.
authors: Department of Anatomy and Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway.
authors: Max
authors: M
authors: Larsson
authors: Cecilie
authors: C
authors: Morland
authors: Irais
authors: I
authors: Poblete-Naredo
authors: Jürg
authors: J
authors: Biber
authors: Niels Christian
authors: NC
authors: Danbolt
authors: Vidar
authors: V
authors: Gundersen
references: Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5979-83
references: 8327470
references: Am J Physiol. 1994 May;266(5 Pt 2):F767-74
references: 7515582
references: Neuroscience. 2005;136(3):649-60
references: 16344142
references: J Comp Neurol. 2003 Oct 13;465(2):161-3
references: 12949777
references: Nature. 2010 Sep 30;467(7315):591-5
references: 20882016
references: Methods Enzymol. 1998;296:388-407
references: 9779462
references: Pflugers Arch. 2004 Feb;447(5):763-7
references: 12750889
references: Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5372-7
references: 9560283
references: Anat Embryol (Berl). 2006 Aug;211(4):257-66
references: 16435108
references: Nat Protoc. 2008;3(1):144-52
references: 18193031
references: Brain Res. 1997 Oct 24;772(1-2):149-55
references: 9406966
references: Cell. 1997 Dec 12;91(6):799-809
references: 9413989