Nuclear and Cytoplasmic Localization of the β-Amyloid Peptide (1–43) in Transfected 293 Cells

• Published in: Biochemical and biophysical research communications 1996-03, Vol.220 (3), p.710-718
• Main Authors: Johnstone, E.M., Babbey, Lael E., Stephenson, Diane, Paul, Donald C., Santerre, Robert F., Clemens, James A., Williams, Daniel C., Little, Sheila P.
• Format: Article
• Language: English
• Subjects: Amyloid beta-Peptides - analysis; Amyloid beta-Peptides - biosynthesis; Amyloid beta-Peptides - genetics; Amyloid beta-Protein Precursor - biosynthesis; Amyloid beta-Protein Precursor - genetics; Animals; Base Sequence; Blotting, Western; Cell Line; Cell Nucleus - metabolism; Cell Nucleus - ultrastructure; Cytoplasm - metabolism; Cytoplasm - ultrastructure; DNA Primers; DNA, Complementary; Exons; Fluorescent Antibody Technique; Genomic Library; Humans; Introns; Kidney; Mammals; Microscopy, Electron; Molecular Sequence Data; Mutagenesis, Insertional; Peptide Fragments - analysis; Peptide Fragments - biosynthesis; Peptide Fragments - genetics; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Sorting Signals - biosynthesis; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Transfection
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1996.0469

Cultures of transformed human embryonic kidney 293 cells were transiently transfected with minigene constructs coding for the Aβ peptide (1–43). The Aβ minigene used in this study consisted of exons 16 and 17 of the amyloid precursor protein gene, including the 6000+ bp intronic region. Two of the constructs used in this study, human amyloid precursor protein (APP) promoter-driven Aβ minigene and BK virus enhancer/adenovirus major late promoter-driven Aβ minigene, did not contain a signal peptide sequence, whereas the third, human APP promoter-signal peptide Aβ minigene did not contain the human APP signal sequence. The resulting Aβproducts were detected by immune precipitation, using 10D5 antibody and Western blot analysis, using R1280 antisera, as SDS stable oligomers in cell lysates of cells containing all three constructs or in culture media when produced by the signal peptide construct. Evaluation of the cells by immunocytochemistry using conventional and transmission electron microscopy indicated that the cells transfected with constructs without the signal peptide accumulated immunoreactive Aβ primarily in the nucleus.