Each line represents an individual bacterial clone that was sequenced

‘s disease. Brain 136: 13831398. Preparation of synthetic A oligomers and fibrils A11-immunoreactive A oligomers were prepared in vitro as previously described. Briefly, 50 g of A was dissolved in 20 L of hexafluoroisopropanol for 15 min at room temperature. The resulting A solution was added to 180 L of ddH2O in a siliconized Eppendorf tube. After 15 min incubation at room temperature, the samples were centrifuged for 15 min at 16 / 26 Characterizing a Model of -Amyloid Toxicity 14,000 g and the supernatant fraction was transferred to a new siliconized tube, and the HFIP was evaporated off. The samples were then stirred using a Teflon coated micro stir bar for 2448 hr at 22C. OC-immunoreactive, soluble A fibrillar aggregates were a kind gift from Dr. Robert Tycko, National Institues of Health, Bethesda, MD. Protein extraction To better characterize protein of interest, we used three different extraction protocols to isolate proteins according to their solubility and Entinostat cellular compartmentalization. A three-step protocol was used to extract brain proteins that were used: 1) to measure levels of total A in rTg9191 mice, 2) to measure levels of water-soluble A dimers in rTg9191 mice, and 3) to quantify soluble oligomers that are immunoreactive to OC or A11 antibodies in rTg9191 and Tg2576 mice and AD PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786681 patients. To prepare the water-soluble fraction, tissue specimens were weighed, transferred to 4 volumes of ice-cold buffer A; and phosphatase inhibitor cocktails ) and homogenized using a Dounce homogenizer. The resulting material was centrifuged for 90 min; the supernatant was then depleted of endogenous immunoglobulins and stored at -20C until further use. The pellets were reserved for the extraction of detergent-soluble proteins. To extract detergent-soluble proteins, the pellets obtained above were transferred to 4 volumes of ice-cold buffer B Triton-X-100; 0.1 mM phenylmethylsulfonyl fluoride; 0.2 mM 1,10-phenanthroline monohydrate; protease inhibitor cocktail; and phosphatase inhibitor cocktails ) and homogenized using a Dounce homogenizer. The resulting material was centrifuged for 90 min; the supernatant was depleted of endogenous immunoglobulins and stored at -20C until further use. The pellets were reserved for the extraction of detergent-insoluble proteins. To extract insoluble proteins, the pellets obtained above were transferred to 40 L of 70% formic acid and homogenized by repeatedly pipetting and vigorously shaking at room temperature for 30 min. The acidic pH of the resulting material was neutralized using 800 L of 1 M Tris-base solution and centrifuged for 90 min. The supernatant was collected PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 and stored at -20C until further use. A four-step fractionation protocol was employed 1) to determine the expression level of human APP, 2) to determine the degree of DOX-mediated suppression of APP expression, and 3) to investigate the regional pattern of APP expression. Each hemi-forebrain was mechanically homogenized in 500 L of Extraction Buffer 1 SDS; and 0.01% NP-40, with the protease and phosphatase inhibitors mentioned above). Supernatants were collected after centrifugation to obtain soluble, extracellular-enriched proteins. The resulting pellets were homogenized in 500 L Extraction Buffer 2 Triton-X-100, with protease and phosphatase inhibitors) followed by centrifugation. Supernatants were collected to obtain cytoplasmic proteins. The resulting pellets were gently agitated in 1 mL Extraction Buffer 3 SDS; 1% deoxycholate; and