Rabbit anti presenilin 1

Protein samples were homogenised in lysis buffer (20 mM Tris, 150 mM NaCl, 1% (v/v) Triton X-100) containing 1 mM PMSF, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, and leupeptin. The samples were then incubated for 30 min at 4 °C and centrifuged for 10 min at 13,000 × g. The supernatant (lysate) was collected, and 15 μg protein was loaded into each lane. Samples were separated on 8–12% SDS-polyacrylamide gels and electro-blotted onto nitrocellulose membranes (Millipore). After blocking with 5% (w/v) non-fat milk, the blots were incubated with the following primary antibodies: rabbit anti-phospho-Akt (p-Akt-Ser473, 1:400, Santa Cruz), rabbit anti-Akt (1:400, Santa Cruz), rabbit anti-phospho-GSK-3β (p-GSK-3β-Ser9, 1:1000, Cell Signaling Technology), rabbit anti-GSK-3β (1:1000, Cell Signaling Technology), mouse anti-phospho-tau (p-tau-Ser396, 1:1000, Cell Signaling Technology), mouse anti-tau (1:1000, Cell Signaling Technology), rabbit anti-presenilin 1 (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz). Conjugated goat anti-rabbit or goat anti-mouse IgG was detected with enhanced chemiluminescence (ECL) (Pierce^® ECL Western Blotting Substrate). β-actin was used as an internal reference for relative quantification.

After treatment, cells were lysed by RIPA lysis buffer and electrophoresed on SDS-polyacrylamide gel with a loading volume of 1 µg/µL. Proteins (10 μg) were separated onto 4–12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Then membranes were incubated in 5% BSA solution at room temperature for 1 h. Subsequently, the membranes were respectively incubated with primary antibodies: rabbit-anti-APP C-terminal (A8717, Sigma-Aldrich, Burlington, MA, USA), rabbit-anti-BACE1 (ab108394, Abcam, Cambridge, UK), rabbit-anti-Presenilin 1 (5643, Cell Signaling Technology, Danvers, MA, USA), rabbit-anti-Presenilin 2 (9979, Cell Signaling Technology, Danvers, MA, USA), mouse-anti-Human sAPPβ-sw (10,321, IBL, Minneapolis, MN, USA), rabbit-anti-APH1 (AB9214, Millipore, Burlington, MA, USA), mouse-anti-β-Actin (A1987, Sigma-Aldrich, Burlington, MA, USA) overnight at 4 °C. Membranes were washed with TBST and subsequently incubated with the horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibodies (1:5000) for 1 h at room temperature. The bands were processed with ECL kit (WBKLS0500, Millipore, Burlington, MA, USA) and observed in the chemiluminescence imaging system (ChemiOoc XRS+, Bio-Rad, Hercules, CA, USA). Protein intensities were semi-quantitatively analyzed using the NIH ImageJ software [66 (link),69 (link)].