Anti nicastrin

Soluble fraction of brain lysates was used for western blot. Fifteen to thirty micrograms of brain lysates were separated by SDS-PAGE on SDS gels (5% stacking, 10~15% resolving) and transferred to nitrocellulose membranes (110V constant for 90min, wet transfer). Some of the protein samples were separated on Bolt^TM 4–12% Bis-Tris Plus gels with MES running buffer (200 V constant, for 25 min) and transferred to nitrocellulose membranes using Bio-Rad wet transfer system (100 V constant for 1 h). The membranes were blocked with 5% skim milk in TBST (200mM Tris, 3M NaCl, 1% TWEEN-20, pH 7.6). The membranes were labeled with primary antibodies (anti-GAPDH, 1:1000, Bethyl #A300-641A, RRID:AB_513619; anti-PEN2, 1:500 ,Cell Signaling #8598, RRID:AB_11127393; anti-APH-1, 1:100, Thermo Fisher #PA1-2010, RRID:AB_2227105; anti-Nicastrin, 1:1000, Cell Signaling #5665, RRID:AB_10694688; anti-BACE1, 1:1000, Cell Signaling #5606, RRID:AB_1903900; anti-β-amyloid, 1:100, BioLegend #803002, RRID:AB_2564654), for overnight at 4°C and then labeled with the HRP-conjugated secondary antibodies (anti-Rabbit IgG HRP-conjugate, 1:5,000, Promega #W4011, RRID:AB_430833; anti-mouse IgG Secondary antibody HRP, 1:5000, Thermo Fisher #31430, RRID:AB_228307). Chemiluminescence images were captured by ImageQuant LAS 4000 (Fuji, RRID:SCR_014246).

Primary antibodies included anti-Aβ 4G8 (SIG-39220, BioLegend, San Diego, CA, USA), anti-SPIN90 (generated in our laboratory), anti-Rab11 (ab3612, Abcam, Cambridge, UK), anti-GFP (sc-9996, Santa Cruz Biotechnology, Dallas, TX, USA), anti-APP (MAB348, EMD Millipore, Darmstadt, Germany), anti-BACE1 (B0681, Sigma, St. Louis, MO, USA), anti-Nicastrin (5665, Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p35/p25 (2680, Cell Signaling Technology, MA, USA) and anti-tubulin (T6199, Sigma, St. Louis, MO, USA). Secondary antibodies included horseradish peroxidase (HRP)-conjugated donkey anti-mouse (115-035-006) and anti-rabbit antibodies (111-035-006, Jackson Laboratory, Bar Harbor, ME, USA). Unless otherwise noted, all chemicals were purchased from Sigma.