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  • Multiplex assay for live-cell monitoring of cellular fates of amyloid-β precursor protein (APP).

Multiplex assay for live-cell monitoring of cellular fates of amyloid-β precursor protein (APP).

PloS one (2014-06-17)
Maria Merezhko, Pranuthi Muggalla, Niko-Petteri Nykänen, Xu Yan, Prasanna Sakha, Henri J Huttunen
ABSTRACT

Amyloid-β precursor protein (APP) plays a central role in pathogenesis of Alzheimer's disease. APP has a short half-life and undergoes complex proteolytic processing that is highly responsive to various stimuli such as changes in cellular lipid or energy homeostasis. Cellular trafficking of APP is controlled by its large protein interactome, including dozens of cytosolic adaptor proteins, and also by interactions with lipids. Currently, cellular regulation of APP is mostly studied based on appearance of APP-derived proteolytic fragments to conditioned media and cellular extracts. Here, we have developed a novel live-cell assay system based on several indirect measures that reflect altered APP trafficking and processing in cells. Protein-fragment complementation assay technology for detection of APP-BACE1 protein-protein interaction forms the core of the new assay. In a multiplex form, the assay can measure four endpoints: total cellular APP level, total secreted sAPP level in media, APP-BACE1 interaction in cells and in exosomes released by the cells. Functional validation of the assay with pharmacological and genetic tools revealed distinct patterns of cellular fates of APP, with immediate mechanistic implications. This new technology will facilitate functional genomics studies of late-onset Alzheimer's disease, drug discovery efforts targeting APP and characterization of the physiological functions of APP and its proteolytic fragments.

MATERIALS
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Sigma-Aldrich
Anti-Amyloid Precursor Protein, C-Terminal antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
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25 μL
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A recombinant, preservative-free antibody is available for your target. Try ZRB1151
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100 μL
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A recombinant, preservative-free antibody is available for your target. Try ZRB1151
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0.2 mL
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A recombinant, preservative-free antibody is available for your target. Try ZRB1151
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$555.96