Anti p35p25

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.

For reducing SDS‒PAGE analysis, cell or brain lysates were mixed with 4x Laemmli buffer containing 10% β-mercaptoethanol (BME) and boiled at 97 °C for 5 min. For nonreducing SDS‒PAGE analysis, cell or brain lysates were mixed with 4x Laemmli buffer without BME. For immunoblot analysis, 10 μg of each lysate was separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane. The levels of total tau and phosphorylated tau were detected by anti-tau antibodies against 2B11 (IBL), Tau5 (Abcam), pSer199 (Abcam), pSer396 (Abcam), and pThr205 (Abcam). For immunoblot analysis of tau kinases, the levels of total and phosphorylated tau kinase were detected by anti-GSK3β (Abcam), anti-ERK1/2 (Cell Signaling), anti-P35/P25 (Cell Signaling), and anti-CDK5 (Abcam) antibodies. β-actin (Abcam) and GAPDH (Cell Signaling) were used as loading controls. Band intensity was quantified using ImageJ software (NIH). All data were normalized to β-actin.