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  • Title: Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage.
    Author: Dmitriev AD, Factor MI, Segal OL, Pavlova EV, Massino YS, Smirnova MB, Yakovleva DA, Dmitriev DA, Kizim EA, Kolyaskina GI, Brusov OS.
    Journal: Clin Chim Acta; 2005 Jun; 356(1-2):76-94. PubMed ID: 15936305.
    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.
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