App 6e10

The following antibodies were used: β3 tubulin (Santa Cruz Biotechnology, Dallas, TX, USA, #80016), Actin (Sigma-Aldrich, St. Louis, Missouri, USA, #A4700), APP 6E10 (BioLegend, San Diego, CA, USA, #803001), APP Y188 (Abcam, Cambridge, UK, #ab32136), Drp1 (BD Bioscience, San Jose, CA, USA, #611112), GAPDH (Enzo LifeScience, Exeter, UK, #ADI-CSA-335-E), IP3R3 (BD Biosciences, San Jose, CA, USA, #610312), LC3B (Cell Signalling, Danvers, MA, USA, #3868; Novus Biologicals, Centennial, CO, EUA, #NB100-2220), Mfn1 (Santa Cruz Biotechnology, #SC50300), Mfn2 (Abcam, #Ab56889), Opa1 (BD Bioscience, San Diego, CA, USA, #612606), SQSTM1/p62 (Cell Signalling,#5114), TIM23 (BD Biosciences, #611223), TOM20 (Santa Cruz Biotechnology, #sc-11415), TOM70 (Santa Cruz Biotechnology, #sc-366282) and VDAC1 (Abcam, #Ab14734).

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.