Nuclear and cytoplasmic localization of the beta-amyloid peptide (1-43) in transfected 293 cells.
Cultures of transformed human embryonic kidney 293 cells were transiently transfected with minigene constructs coding for the Abeta peptide (1-43). The Abeta 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 Abeta minigene and BK virus enhancer/adenovirus major late promoter-driven Abeta minigene, did not contain a signal peptide sequence, whereas the third, human APP promoter-signal peptide Abeta minigene did not contain the human APP signal sequence. The resulting Abeta products 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 Abeta primarily in the nucleus.
Johnstone
E M
EM
Central Nervous System/GI/GU/Molecular Biology Research Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Babbey
L E
LE
Stephenson
D
D
Paul
D C
DC
Santerre
R F
RF
Clemens
J A
JA
Williams
D C
DC
Little
S P
SP
D016229:Amyloid beta-Peptides / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D017931:DNA Primers; D018076:DNA, Complementary; D005091:Exons; D005455:Fluorescent Antibody Technique; D015698:Genomic Library; D006801:Humans; D007438:Introns; D007668:Kidney; D008322:Mammals; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D014162:Transfection
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C098994:amyloid beta-protein (1-43)