Detailed Immunoblotting and Microscopy Protocol

For confocal microscopy, we used the rat monoclonal anti-LAMP1 (sc-19992, Santa Cruz, 1:200). For WB, the following antibodies were used: mouse monoclonal anti-NCT (Esselens et al., 2004 (link)) (9C3, 1:7000), rabbit polyclonal anti-PSEN1-NTF (ab71181, Abcam, 1:2000) or rat polyclonal anti-PSEN1-NTF for co-immunoprecipitation (MAB1563, Millipore, 1:4000), rabbit monoclonal anti-PSEN1-CTF (ab76083, Abcam, 1:2000), rabbit polyclonal anti-PEN2 (ab18189, Abcam, 1:1000), rabbit polyclonal anti-APP-CTF (Esselens et al., 2004 (link)) (B63, 1:10,000) and mouse monoclonal anti-transferrin receptor (136800, Invitrogen, 1:4000). Secondary antibodies included HRP-conjugated goat-anti-mouse and goat-anti-rabbit (Bio-Rad, 1:10000 dilution). Co-immunoprecipitation was done with rabbit anti-GFP (#A11122; Invitrogen). For quantitative WB, mouse monoclonal anti-PSEN1-CTF (MAB5232, Millipore, 1:1000 dilution) and near-infrared goat anti-mouse Alexa Fluor790 (#A11375, Invitrogen, 1:15,000 dilution) were used. For PM sheet immunofluorescence, GFPbooster-Atto647N (Chromotek, 1:1000 dilution) was used.

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Escamilla-Ayala A.A., Sannerud R., Mondin M., Poersch K., Vermeire W., Paparelli L., Berlage C., Koenig M., Chavez-Gutierrez L., Ulbrich M.H., Munck S., Mizuno H, & Annaert W. (2020). Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface. eLife, 9, e56679.

Publication 2020

sc-19992 MAB1563 ab76083 ab18189 MAB5232 GFPbooster-Atto647N

Antibodies Co immunoprecipitation Confocal microscopy Dilution Goat Immunofluorescence Lamp1 Mouse Psen1 Rabbit Transferrin receptor

Corresponding Organization : KU Leuven

Other organizations : Université de Bordeaux, Inserm, Centre National de la Recherche Scientifique, Bordeaux Population Health, University of Freiburg, VIB-KU Leuven Center for Microbiology, Einstein Center for Neurosciences Berlin, Charité - Universitätsmedizin Berlin, PicoQuant (Germany), University Medical Center Freiburg

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Variable analysis

independent variables

Antibody dilutions for immunofluorescence and Western blotting

dependent variables

Protein expression and localization

control variables

Antibody sources and catalog numbers
Secondary antibody sources and dilutions

controls

Positive control: No specific positive controls mentioned.
Negative control: No specific negative controls mentioned.

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