Quantitative Western Blot Analysis of Hippocampal Proteins

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.