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Clin Chim Acta. 2005 Jun;356(1-2):76-94. Epub 2005 Mar 17.

Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage.

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  • 1Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117485 Russia. dad75@rol.ru

Abstract

BACKGROUND:

Serotonin transporter (SERT) is important target molecule for many antidepressive drugs and substances of abuse and is implicated in psychiatric disorders. We performed immunoblotting analysis of human and rat SERT in platelets and brain using the panel of eight site-specific SERT monoclonal and polyclonal antibodies (mAbs and pAbs).

METHODS:

SDS-PAGE/Western blotting was conducted using peroxidase-labeled DEAE and affinity purified SERT antibodies under conditions preventing SERT post-extraction degradation.

RESULTS:

Immunoreactive polypeptides of 14, 22, 32, 35, 37, 56, 68, and approximately 150-200 kDa were revealed in human platelet extracts using N-terminal and C-terminal SERT antibodies. In rat brain, C-terminal mAbs detected 68, 56, and 37 kDa proteins, in postmortem human brain predominated 35-37 kDa proteins. The immunoreactivity was abolished after antibody preadsorption with antigens. N-terminal pAbs recognized the 68 kDa protein, affinity purified on C-terminal mAbs, confirming its identity as full-size human SERT (the predicted size approximately 70.5 kDa).

CONCLUSIONS:

The explanation of the results of immunoblotting most likely is a site-specific SERT endoproteolytic cleavage and a marked difference in glycosylation rather than nonspecific protein degradation, cross-reactivity with other epitopes or SERT alternative splicing.

PMID:
15936305
[PubMed - indexed for MEDLINE]
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