Antipeptide antibodies confirm the topology of the human norepinephrine transporter

M. Bruss, R. Hammermann, S. Brimijoin, H. Bonisch

Research output: Contribution to journalArticlepeer-review

67 Scopus citations


We have raised polyclonal antibodies (N6-28, L211-226, L371-384, and C590- 607) against peptides corresponding to hydrophilic sequences of the human norepinephrine transporter (hNET). The antisera immunoprecipitated the [35S]Met-labeled hNET. Antiserum L211-226, directed against a sequence of the putative second (large) extracellular loop of hNET, also immunoprecipitated the human dopamine transporter. Antisera N6-28 and C590- 607, raised against a hNET peptide region of the N and the C termini, respectively, recognized a 58-kDa protein from transfected COS-7 cells expressing the hNET. This 58-kDa species represents a functional, glycosylated form of the hNET and not a degradation product. Tunicamycin treatment of transfected COS-7 cells as well as peptide-N-glycosidase F digestion of the transporter converted the 58-kDa species to a 50-kDa form, indicating that the latter represents the hNET core protein. In indirect immunofluorescence studies, our antisera confirmed the originally proposed topology of hNET. Antisera N6-28 and C590-607 detected hNET only in permeabilized cells. In contrast, antisera L211-226 and L371-384 directed against peptide sequences of the second and fourth putative extracellular loop displayed fluorescence signals with the intact cells.

Original languageEnglish (US)
Pages (from-to)9197-9201
Number of pages5
JournalJournal of Biological Chemistry
Issue number16
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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