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Additional info for Amyloid Precursor Protein A Practical Approach
2. Weidemann, A. et al. Identification, biogenesis, and localization of precursors of Alzheimer’s disease A4 amyloid protein. Cell 57, 115, 1989. 3. Schubert, W. et al. Localization of Alzheimer beta A4 amyloid precursor protein at central and peripheral synaptic sites. Brain Res. 563, 184, 1991. 4. Okamoto, T. et al. Intrinsic signaling function of APP as a novel target of three V642 mutations linked to familial Alzheimer’s disease. EMBO J. 15, 3769, 1996. 5. Zambrano, N. et al. Interaction of the phosphotyrosine interaction/phosphotyrosine binding-related domains of Fe65 with wild-type and mutant Alzheimer’s beta-amyloid precursor proteins.
J. Biol. Chem. 269, 17386, 1994. 2. Nordstedt, C. et al. Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells. J. Biol. Chem. 268, 608, 1993. 3. J. Trafficking of cell-surface amyloid betaprotein precursor. II. Endocytosis, recycling and lysosomal targeting detected by immunolocalization. J. Cell Sci. 109, 999, 1996. 4. Simons, M. et al. Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proc. Natl.
Expressing clones are identified by silver stain sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the culture supernatants. The expressing clones are then grown in culture to produce large amounts of GFD for crystallography and biological assays as described below. 3 METHOD FOR EXPRESSION 1. Shaker flask cultures are grown by inoculating a suitable high-level expression clone into 500 ml YPD in a 2-liter baffled flask. 2. Cultures are grown for 48 hr at 3oC on an orbital shaker (250 rpm) to a cell density between 45 and 60 × 107 cells/ml (optical density of 15 to 20 at 600 nm).