Ar0138

Hippocampal tissues were prepared as described above. Cells (see below) were washed twice with ice-cold PBS. The tissues and cells were homogenized in cold RIPA buffer (AR0102, BOSTER) containing a cocktail of PMSF (AR1179, BOSTER) and protein phosphatase inhibitor (AR1183, BOSTER). The homogenates were then centrifuged (16,000×g, 30 min, 4 °C), and the supernatants were collected. The total protein concentrations of the samples were measured by a BCA Protein Assay Kit (PC0020, Solarbio). Total protein for each sample was diluted 1:1 with loading buffer (AR0131, BOSTER) and boiled for 5 min at 95 °C. Equal amounts of total protein were separated by 12% or 15% SDS-PAGE (AR0138, BOSTER) and transferred to PVDF membranes (0.45 μm or 0.22 μm, Millipore). Following blocking with 5% BSA for 2 h at room temperature, the membranes were incubated with desired primary antibodies overnight at 4 °C, and then HRP-conjugated secondary antibody for another 2 h at room temperature. After several washes, the protein bands were developed with ECL Western Blot Detection kit (P0018FS, Beyotime) and detected using Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems). The band intensity was analyzed with AlphaView SA (Fluorchem FC3, ALPHA).

Cells and tissues were collected and lysated using RIPA buffer (100:1:1 ratio of phosphatase inhibitor to protease inhibitor). After 30 min of lysis on ice, the total protein was extracted. After separation by SDS-PAGE (AR0138, Boster Biotechnology), the protein was transferred onto a nitrocellulose membrane (AR0135-04, Boster Biotechnology). The cells and tissues were then blocked with 5% skim milk at room temperature for 2 h and incubated with an appropriate concentration of primary antibody overnight at 4 °C. Finally, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (H + L) antibody (abs20002ss; Absin Bioscience) at room temperature for 2 h. The immunoreactive bands were detected using western blotting.