Ab241592

Western blot and analysis were carried out, as described previously (Liu et al., 2011 (link)). In brief, proteins (5–10 μg of total protein) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked in 5% skimmed milk. The primary antibodies were rabbit anti-APP (1:2000, ab241592, Abcam), rabbit anti-PSD-95 (1:1000, ab238135, Abcam), or mouse anti-β-actin (1:4000, #3700, CST). The secondary antibodies were corresponding horseradish peroxidase–conjugated ones. A chemiluminescent substrate kit (Thermo Fisher Scientific, United States) was used and developed by using an X-ray film (Kodak) in the dark room. The developed films were scanned, and the percentage of the band relative intensity was analyzed by ImageJ software.

Adherent primary neurons were collected using radioimmunoprecipitation assay buffer, whereas the tissue homogenates of cortical or hippocampal tissues of mice were dissolved in protein lysate (Biyuntian Co., Ltd., P0013C, China). Centrifugation was performed (12000 rpm, 15 min, 4°C), and the supernatant was absorbed as extracted protein.
In all, 40 μg of protein was extracted from each sample and isolated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was placed in a blocking solution to reduce nonspecific binding. Subsequently, the membrane was washed with Tris-buffered saline Tween-20 (TBST).
Following this, the membrane was incubated with GAPDH antibody (1 : 1000, Abcam, Ab9485, UK), APP antibody (1 : 1000, Abcam, Ab241592, UK), Navβ2 antibody (1 : 1000, Abcam, Ab138064, UK), BDNF antibody (1 : 1000, Abcam, Ab108319, UK), or NEP antibody (1 : 1000, R&D Systems, AF1182, USA) at 4°C overnight. After washing with 1x TBST, the membrane was incubated with secondary antibodies (1 : 1000, Abbkine, A23220/A23410, USA) at room temperature for 1 h and rinsed with 1x TBST again. All results were photographed using Bio-Rad Gel Imaging System (ChemiDoc™ XRS+). ImageJ (National Institutes of Health, USA) was used for strip quantitative analysis.