Rabbit anti bace1

Hippocampus tissues were removed and homogenized in neuronal Protein Extraction Reagent (Thermo Fisher Scientific, 87792) containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, 87786). Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, 23227). Equal amounts of total protein were separated in 12% SDS–PAGE gels and then transferred to nitrocellulose (NC) membranes (Solarbio, Beijing, China). The primary antibodies were: rabbit anti-ADH1B (Biorbyt, 1:800), rabbit anti-BACE 1 (Abcam, 1:1000), rabbit anti-IDE (Abcam, 1:1000), rabbit anti-p75NTR (Cell Signaling Technology, 1:1000), rabbit anti-cleaved caspase-3 (Abcam, 1:1000), rabbit anti-Bcl-2 (Abcam, 1:1000), and rabbit anti-Bax (Abcam, 1:1000). Image Lab (Bio-Rad) was utilized for protein signal densitometry. Detection of proteins from pretreated SH-SY5Y cells using western blotting were performed as previously described (Zhang et al., 2016 (link)).

N2a/APP695 cells (1 × 10⁵ cells/mL) were cultured in a 6-well plate for 24 hours and were then treated with RJPs for 24 hours. Cells were washed with cold PBS and lysed with 1 × loading buffer (10 mM Tris, pH 6.8; 1%SDS; 5% glycerin; 0.1 M DTT; bromophenol blue; 1 mM AEBSF (Sigma, USA)). The lysate was collected and boiled at 100 °C for 10 min. Subsequently, an equal volume of sample was electrophoretically separated using 10% SDS-PAGE and then transferred to an NC membrane (Millipore, USA). Membranes were incubated overnight at 4 °C with various primary antibodies. The primary antibodies used in this study: mouseanti-APP (6E10, 1:250, Covance, USA), rabbit-anti-BACE1 (1:500, Abcam, USA), rabbit-anti-HDAC1 (1:5000, AB clonal, Wuhan, China), rabbit-anti-β-actin and rabbit-anti-GAPDH (1:2000, Cell Signaling Technology, USA). The secondary antibodies used were goat-anti-mouse IgG IRDye 800CW (1:10000, Li-COR, USA) and goat-anti-rabbit IgG IRDye 800CW (1:10000, Li-COR, USA). After incubation with the secondary antibody, the membranes were washed 4 times for 5 min each time. Finally, we used the LI-COR Odyssey Infrared Fluorescence Scanning System (Li-COR, USA) to quantify the fluorescence intensity. The intensity of the bands was analyzed using the system software.

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)].