High binding assay black plates

The cells were cultured on 96-well plates (Corning) overnight then transfected with pcDNA-TTR or control plasmid. The culture medium was collected after 24 hours. The medium was analyzed by an Aβ ELISA as follows. Monoclonal 6E10 anti-Aβ(residues 1–17) mouse IgG1, (Biolegend) was coated in 50 mm carbonate buffer, pH 9.6, at 4°C overnight on high binding assay black plates (Costar), washed with TBST (tris buffered saline with 0.05% Tween 20) and blocked with 5% non-fat milk in TBST. Samples and standards (synthetic Aβ_1-40) were incubated for 2 hr, followed by addition of biotin-labeled 4G8 [anti-Aβ residues 17–24, mouse IgG2b (Biolegend)] and incubation for 1 hr at 37°C. After washing, streptavidin horseradish peroxidase (HRP) conjugate (Invitrogen) was added and incubated for 45 min, followed by detection by Quanta Blue fluorogenic peroxidase substrate (Thermo Scientific) using a Tecan Safire II fluorescence plate reader.

7PA2 or 7WD10 cells were cultured on 96-well plates (Corning) and treated with IRE1 activating compounds +/− 4μ8c overnight. The medium was then replaced with fresh medium containing treatment at a reduced volume (50%), culture medium was collected after 24 hrs. The medium was analyzed by an Aβ ELISA as follows. Monoclonal 6E10 anti-Aβ(residues 1–17) mouse IgG1, (Biolegend) was coated in 50 mm carbonate buffer, pH 9.6, at 4°C overnight on high binding assay black plates (Costar), washed with TBST (tris buffered saline with 0.05% Tween 20) and blocked with 5% non-fat milk in TBST. Samples and standards (condition 7PA2 media) were incubated for 1.5 hrs, followed by addition of 4G8 antibody [anti-Aβ residues 17–24, mouse IgG2b (Biolegend)] conjugated to horseradish peroxidase (HRP) and incubated for 1.5 hrs at 25°C. After washing, ABTS substrate was added, followed by detection with an absorbance plate reader.