Mab5232

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.

Microsomal membranes from native HEK293 cells were pelleted and homogenized in 25 mM HEPES pH 7.4, 1 mM EDTA, 0.25 M sucrose, complete protease inhibitor cocktail (Roche); centrifuged at 3,000 × g for 10 min; and the supernatant was then centrifuged at 100,000 × g for 1 hr. The microsomal membrane was preincubated with 1% DMSO or 10 pM–100 nM of compound E overnight. Equal amounts of microsomal membranes were solubilized in 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, complete protease inhibitor cocktail with 0.1% (w/v) dodelcyl maltoside (DDM; Affimetrix) for 1 hr. The homogenates were centrifuged at 100,000 × g for 30 min, and the supernatant was collected. DDM was added to final DDM concentrations of 0.5%, 1%, and 1.5%. Membrane lysates were incubated overnight at 4°C with anti-nicastrin antibody and Protein G-Sepharose (GE Healthcare). After washing, the captured proteins were eluted with 1× sample buffer (lithium dodecyl sulfate [LDS]; Invitrogen). Samples were resolved on 12% Bis-Tris NuPAGE gels (Invitrogen), transferred onto polyvinylidene fluoride (PVDF) membranes, and probed with anti-nicastrin, PS1-NTF, aph1 (Covance PRB-550P), PS1-CTF (Chemicon MAB5232), and pen2 (Sigma P5622).