Comparison of Anti-Shadoo Antibodies – Where is the Endogenous Shadoo protein?

Authors: Eszter Tóth, Ervin Welker

Abstract:

Shadoo protein (Sho) was described in 2003 as the newest member of Prion protein superfamily [1]. Sho has similar structural motifs like prion protein (PrP) that is known for its central role in transmissible spongiform enchephalopathies. Although a great number of functions have been proposed, the exact physiological function of PrP is not known yet. Investigation of the function and localization of Sho may help us to understand the function of the Prion protein superfamily. Analyzing the subcellular localization of YFP-tagged forms of Sho, we detected the protein in the plasma membrane and in the nucleus of various cell lines. To reveal the localization of the endogenous protein we generated antibodies against Shadoo as well as employed commercially available anti-Shadoo antibodies: i) EG62 anti-mouse Shadoo antibody generated by Eurogentec Ltd.; ii) S-12 anti-human Shadoo antibody by Santa Cruz Biotechnology Inc.; iii) R-12 anti-mouse Shadoo antibody by Santa Cruz Biotechnology Inc.; iv) SPRN antibody against human Shadoo by Abgent Inc. We carried out immunocytochemistry on non-transfected HeLa, Zpl 2-1, Zw 3-5, GT1-1, GT1-7 and SHSY5Y cells as well as on YFP-Sho, Sho-YFP, and YFP-GPI transfected HeLa cells. Their specificity (in antibody-peptide competition assay) and co-localization (with the YFP signal) were assessed.

Keywords: Shadoo, prion protein, immunocytochemistry, antibody-peptide competition assay, antibody.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1074541

References:

[1] M. Premzl, Shadoo, a new protein highly conserved from fish to mammals with similarity to prion protein, Gene, 2003, 314:89-102
[2] B. Caughey, Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders, Annu Rev Neurosci, 2003, 26:267-298
[3] E. T├│th, The hightly-conserved, N-terminal (RXXX)8 motif of mouse Shadoo mediates nuclear accumulation, BBA Mol Cell Res, submitted for publication