Hippocampal Protein Analysis Using Western Blot

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)).