1887

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Volume 97, Issue 8

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrP-specific immunostaining Free

Abstract

We established abnormal isoform of prion protein (PrP)-specific double immunostaining using mAb 132, which recognizes aa 119–127 of the PrP molecule, and novel PrP-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrP-specific double immunostaining, we analysed PrP in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrP-specific double immunostaining showed the existence of PrP positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrP species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrP staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrP stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrP stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrP in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrP in N2a-3 lack the N-terminal portion. In contrast, nearly half PrP detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrP that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrP-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

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Keyword(s): guanidine isothiocyanate , Immunofluorescence assay , monoclonal antibody , primary neurons and prions
© 2016 The Authors
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2016-08-01
2024-04-16
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Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining
J. Gen. Virol. 97, 2030 (2016); https://doi.org/10.1099/jgv.0.000514
/content/journal/jgv/10.1099/jgv.0.000514
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