β actin

The proteins were loaded on 8–10% Tris-glycine SDS-PAGE gel. Proteins were transferred to 0.2-μm nitrocellulose membrane and the transferred membrane was blocked with 5% (w/v) nonfat dried milk in Tris-buffered saline with 0.1% Tween-20 (TBST; 10 mM Tris, 150 mM NaCl, pH 7.6) for 1 h at room temperature. After washing blocked membrane with PBS four times for 10 min, the membrane was incubated with the following primary antibodies at 4 °C for 16 h: APP (6E10; BioLegend, monoclonal, #803002), caveolin (cav-1; BD Transduction Laboratories, polyclonal, #610059), flotillin-1 (BD Transduction Laboratories, monoclonal, #610880), GAPDH (Cell Signaling Technology, Danvers, MA, USA, monoclonal, #14C10), β-actin (EnoGene, New York, NY, USA, monoclonal, #E12-041). Next, the membrane was washed with TBST four times and incubated with horseradish-peroxidase-conjugated goat antirabbit IgG (Invitrogen, polyclonal, #656120) or goat antimouse IgG (Invitrogen, polyclonal, #G21040) antibodies for 1 h at room temperature to detect each primary antibody. After incubation with the secondary antibody, the membrane was washed again with TBST four times. We used enhanced chemiluminescence reagent (Westsave, #LF-QC0101), and signals were captured with film (MTC Bio, Metuchen, NJ, USA, #A8815). The intensity of bands was captured by LAS-3000 system (Fuji Film) and analyzed by Multi Gauge V3.0.

The proteins were loaded on 10% Tris-glycine SDS-PAGE gel and transferred to 0.2 μm nitrocellulose membrane. The transferred membrane was blocked with 5% (w/v) non-fat dried milk in Tris-buffered saline with 1% Tween-20 (TBST) for 1 h at room temperature. After washing blocked membrane with PBS four times for 10 min each, the membrane was incubated with the following primary antibodies: APP (1:2000, 6E10, BioLegend, monoclonal, RRID: AB_2564652; 1:2000, 22C11, Invitrogen, monoclonal, RRID: AB_2572978), caveolin (1:10,000, BD Transduction Laboratories, polyclonal, RRID: AB_397471), BACE1 (1:2000, abcam, RRID: AB_10861218), nicastrin (1:1000, CHEMICON, RRID: AB_2235791) and β-actin (1:10,000, EnoGene, monoclonal, #E12-041) at 4 °C for 16 h. Next, the membrane was washed with TBST four times and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Invitrogen, polyclonal, RRID: AB_2533967) or goat anti-mouse IgG (1:2000, Invitrogen, polyclonal, RRID: AB_2536527) antibodies for 1 h at room temperature to detect each primary antibody. After incubation with secondary antibodies, the membrane was washed again with TBST four times. We used an enhanced chemiluminescence reagent (Westsave, #LF-QC0101), and signals were captured with film (MTC Bio, #A8815). The intensity of bands was measured by the LAS-3000 system (Fuji Film) and analyzed by Multi Gauge V3.0.