0.5% Triton-X-100, and protease and phosphatase inhibitors); subsequent 17 / 26 Characterizing a Model of -Amyloid Toxicity centrifugation yielded a supernatant enriched in membrane-associated proteins and a pellet containing detergent-insoluble proteins. The three detergent-soluble fractions were then depleted of endogenous immunoglobulins. Detergent-insoluble pellets were incubated with 40 L of 70% formic acid, mechanically dissociated, gently agitated, and neutralized with 800 L of 1 M Tris-base solution. Samples were centrifuged and the supernatants were collected and concentrated to ~350400 L/sample using a vacuum concentrator. Protein extracts were stored at -20C until further use. Finally, material obtained from a two-step brain protein extraction protocol was used 1) to measure levels of A 56 in rTg9191, TgArc6, and hAPP-J20 mice, and 2) to determine levels of CTF in rTg9191 and Tg2576 mice. Forebrains were mechanically homogenized in 500 L of extraction buffer SDS; 0.5% Triton-X-100; 1% deoxycholate; and protease and phosphatase inhibitors described above). Homogenates were gently agitated at 
4C for 1 hr, and then centrifuged for 90 min. The resulting supernatant was depleted of endogenous immunoglobulins and stored at -20C until further use. Pellets were extracted using 40 L of 70% formic acid and then neutralized with 400 L of 1 M Tris-base solution. Samples were then centrifuged; supernatants were collected and stored at -20C until further use. Protein concentrations of brain extracts were determined using a BCA protein assay kit according to the manufacturer’s instructions. Immunoblot Western blotting. Western blotting was performed using the protocol of Liu et al.. Proteins were denatured by heating under reducing conditions. Tricine loading buffer glycerol; 8% SDS; 0.01% Coomassie Brilliant Blue; 0.1% Phenol Red; 5% -mercaptoethanol) was added to each sample, and the samples were heated with agitation at 95C for 5 min. The denatured samples were loaded onto pre-cast 1020% SDS-polyacrylamide Tris-Tricine gels and electrophoretically separated at room temperature at constant voltage. The separated proteins were then transferred to nitrocellulose membranes under low temperature conditions. Membranes were then transferred to 50 mL of PBS into 200 mL of ddH2O), and epitopes were retrieved by boiling the membranes twice for 10 sec each time, with a 4 min cooling period after each episode of boiling. Non-specific binding sites were blocked by incubating the membranes on an orbital shaker for 13.5 hr at room temperature in MedChemExpress Triptolide blocking buffer Tween-20; 33% casein blocking buffer; 0.05% cold water fish PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785045 skin gelatin; 5% bovine serum albumin ). Primary antibodies were then added directly to the blocking buffer as follows: mouse monoclonal 22C11, 1:2,000; mouse monoclonal 4G8, 1:10,000; mouse monoclonal 6E10, 1:2,500; biotinylated 6E10, 1:2,500; mouse monoclonal LN27, 1:2,500; mouse monoclonal anti–tubulin, 1:200,000. Membranes 18 / 26 Characterizing a Model of -Amyloid Toxicity were incubated overnight at 4C on an orbital shaker. Following primary antibody incubation, membranes were rinsed once briefly with TBST wash buffer Tween-20) at room temperature, and then PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 an additional five times for 5 min each on the orbital shaker. Membranes were then incubated for 1 hr at room temperature with secondary antibody -conjugated goat-anti-mouse IgG, diluted 1:200,000 in TBST wash buffer; for Fig 6A upper panel, biotin-conjugated goat-anti-mouse I
