Tnt 1

Preparation of brain homogenates, Western blot (WB) and Dot blot (DB) analysis were performed as previously described^{30 (link),62 (link)}. Briefly, 0.2 g of brain tissue from four different AD cases were homogenized in 0.4 ml TBS buffer with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X, Thermo Scientific, CA), then centrifuged at 6400xg for 15 minutes at +4 °C. Supernatants (soluble fractions) were collected and stored at −80 °C for further analysis. For WB soluble fractions applied to electrophoresis on NuPAGE 4–12% Bis-Tris gel in MES buffer under reducing conditions (Invitrogen, CA) and electrotransferred onto nitrocellulose membrane (GE Healthcare, NJ). Tau were visualized by incubating with sera (dilution at 1:1000) from mice immunized with AV-1980R/A and injected with Advax^CpG only followed by HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology, CA). For DB assay the same extracts were applied to membrane (1 μg). Proteins were detected using sera from mice immunized with AV-1980R/A and control mice injected with Advax^CpG only, TNT-1 (Millipore, MA), HT7 (Life Technology, CA) antibodies. All primary antibodies were used at concentration of 1 μg/ml, serum was used at dilution 1:2500. Bovine anti-mouse HRP-conjugated secondary antibody was used (Santa Cruz Biotechnology, CA).

Soluble fractions of brain extracts from AD cases and controls were applied to membrane (2 μg of total protein in 1 μl volume) and proteins were detected using mouse 1C9, TNT-1 (Millipore, MA), HT7 (Life Technology, CA) monoclonal and rabbit polyclonal anti-Tau antibodies (BioLegend, CA). All primary antibodies were used at concentration of 1 μg/ml. Bovine anti-mouse and mouse anti-rabbit HRP-conjugated secondary antibodies were used at concentration of 0.2 μg/ml (Santa Cruz Biotechnology, CA). Proteins were visualized with enhanced chemiluminescence detection using Luminol reagent (Santa Cruz Biotechnology, CA).

Western Blot analyses of recombinant full-length 4R/0 N tau, tauΔ2–18 and soluble fractions from brain homogenates were performed to confirm the specificity and binding ability of 1C9 and Armanezumab to pathological human tau. Commercial HT7 and TNT-1 antibodies have been used as positive controls. Briefly, brain homogenates containing equal amounts of total protein in SDS sample buffer (non-reducing conditions without heat incubation) were subjected to electrophoresis in 10% Bis-Tris polyacrylamide gel in MES buffer (Invitrogen, CA), then electro-transferred onto nitrocellulose membrane (GE Healthcare, NJ). The membranes were blocked overnight with 5% fat-free dry milk in TBS with 0.05% Tween following by detection of tau using 1C9, Armanezumab, HT7 (Life Technology, CA) or TNT-1 (Millipore, MA) monoclonal antibody and appropriate HRP-conjugated secondary antibody. All primary antibodies were used at concentration 1 μg/ml. Proteins were visualized with enhanced chemiluminescence detection using Luminol reagent (Santa Cruz Biotechnology, CA).