Antipeptide Antibodies Confirm the Topology of the Human Norepinephrine Transporter

• DOI: 10.1074/jbc.270.16.9197
• Publication Date: 2002-07-26T14:53:54Z
• Authors: Michael Brüss; Rainer Hammermann; Stephen Brimijoin; Heinz Bönisch
• Keywords: Immunoprecipitation; Polyclonal antibodies; Norepinephrine transporter

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 [³⁵S]Met-labeled hNET. Antiserum directed a sequence putative second (large) extracellular loop hNET, also dopamine transporter. Antisera N6-28 C590-607, hNET peptide region N C termini, respectively, recognized 58-kDa protein from transfected COS-7 cells expressing This species represents functional, glycosylated form not degradation product. Tunicamycin treatment as well peptide- <i>N</i>-glycosidase F digestion converted 50-kDa form, indicating that latter core protein. In indirect immunofluorescence studies, our confirmed originally proposed topology C590-607 detected only in permeabilized cells. contrast, L211-226 L371-384 fourth displayed fluorescence signals with intact