Bace1

Proteins were extracted from hippocampal tissues and quantified according to our previously described method [93 (link)]. After SDS-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to a nitrocellulose (NC) membrane and blocked with 3% bovine serum albumin (BSA). The membrane was incubated with rabbit β-actin (1:1000; Cell Signaling Technology, USA), RFP (1:1000; Abcam, UK), PSD-95, GluA1, GluA2, GluN1, GRIN2A, GRIN2B, BACE1, ADAM10, Nicastrin, APP, or GFP antibody (1:200; Bioss, China) at 4 °C overnight. The membrane was washed with PBS, incubated with IR Dye 800CW-conjugated secondary antibody (1:5000; LiCor Biosciences, USA), and subsequently detected with a LI-COR Odyssey Infrared Fluorescent System. The density of each band was quantified using Image-pro Express software, version 6.0 (Media Cybernetics, USA) and was normalized to the corresponding β-actin value to account for variations in loading.

The homogenized brain tissues were eluted by boiling in SDS-sample buffer. Then, we performed SDS-PAGE to assess brain proteins using the Bio-Rad mini gel system (Vom Berg et al., 2012 (link)). In particular, Aβ and APPct levels were measured with urea-based electrophoresis, and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then probed with antibodies at the appropriate dilutions, including Aβ and full-length APP (6E10, Covance) and APPct (A8717, Sigma–Aldrich) at 1:1000 (Sarajarvi et al., 2009 (link); Heneka et al., 2013 (link)), ADAM10 (Bioss), BACE1 (Bioss), and PS1 (Bioss) at 1:500. We used β-actin (Abcam) as internal controls and Image-Pro Plus software for densitometric analysis.