PSEN1/2 dKO HEK293 cells30 (link) co-transfected with C99–720 and WT or FAD-mutant PSEN1 were fixed with 4% paraformaldehyde (PFA) (VWR, Radnor, PA), and permeabilized by 0.1% Triton-X100 (Sigma-Aldrich, St. Louis, MO). The permeabilized cells were then incubated with mouse monoclonal 6E10 (BioLegend, San Diego, CA) and rabbit polyclonal nicastrin antibodies (Novus Biologicals, LLC, Centennial, CO), followed by an Alexa Fluor™ 488 (FRET donor) or Cy3 (acceptor)-conjugated anti-mouse and rabbit IgG secondary antibodies, respectively (ThermoFisher Scientific). For control experiment in Figure S7 , primary antibody to PSEN1 Loop (EP2000Y; Abcam) was used in place of anti-nicastrin antibody. The slide was covered by a coverslip using Fluoromount-G™ Mounting Medium (ThermoFisher Scientific) and stored at 4 °C.
Mouse monoclonal 6e10
Manufactured by BioLegend
Mouse monoclonal 6E10 is a laboratory reagent used for research purposes. It is a mouse-derived monoclonal antibody that can be used to detect and study specific target proteins or molecules in various experimental applications.
Automatically generated - may contain errors
2 protocols using mouse monoclonal 6e10
1
PSEN1/2 dKO HEK293 Cell FRET Assay
2
APP-derived Fragments Quantification
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For APP-derived fragments (C99 and APP) analysis, the parieto-temporal cortex extracts used for determining Aβ levels were mixed with 4x XT sample buffer (Bio-Rad), boiled for 5 min at 95 ºC, resolved onto a Criterion TM XT precast gel 4-12% Bis-Tris (Bio-Rad) using XT MES (Bio-Rad) running buffer and transferred to nitrocellulose membranes.
In the rest of cases, protein samples were mixed with 6x Laemmli sample buffer, boiled for 5 min at 95 ºC, resolved onto SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
In all cases, membranes were then blocked with 5% milk in TBS and incubated overnight with the following primary antibodies: mouse monoclonal 6E10 (reactive to amino acids 1-17 of Aβ peptide, 1:1000, BioLegend), mouse monoclonal anti-p-Tau One TM software v.4.6.3 (Bio-Rad) was used for protein quantification.
In the rest of cases, protein samples were mixed with 6x Laemmli sample buffer, boiled for 5 min at 95 ºC, resolved onto SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
In all cases, membranes were then blocked with 5% milk in TBS and incubated overnight with the following primary antibodies: mouse monoclonal 6E10 (reactive to amino acids 1-17 of Aβ peptide, 1:1000, BioLegend), mouse monoclonal anti-p-Tau One TM software v.4.6.3 (Bio-Rad) was used for protein quantification.
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