Ab187041

Proteins were extracted from rat hippocampal tissues using radio immunoprecipitation assay buffer (Beyotime) and quantified with a bicinchoninic acid assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts (20 µg) of tissue samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto the polyvinylidene fluoride membranes (Bio‐Rad, Hercules, CA, USA). After being blocked with 5% defatted milk, the membranes were incubated with primary antibodies against: Bax (ab32503, 1:1,000, Abcam), cleaved (C)-caspase3 (#9661, 1:1,000, Cell Signaling, Danvers, MA, USA), phosphorylated (p)-TrkB (ab229908, 1:1,000, Abcam), TrkB (ab187041, 1:5,000, Abcam), p-PI3K (ab182651, 1:1,000, Abcam), PI3K (ab191606, 1:1,000, Abcam), proBDNF (p1374, 1:400, Sigma–Aldrich), mBDNF (ab108319, 1:1,000, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (ab181602, 1:10,000, Abcam) at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab7090, 1:1,000, Abcam) at room temperature for 2 h. Eventually, protein bands were visualized with an enhanced chemiluminescence kit (Bio-Rad), and the intensity values were analyzed with C‐Digit software (LI-COR Biosciences, Lincoln, NE, USA).

Hippocampal tissues were homogenized in RIPA cell lysate (Beyotime, P0013B), centrifuged at 12,000 × g for 15 min, and the supernatant was collected. Each protein sample was loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel spiked wells. Electrophoresis was performed at constant pressure of 80 V for approximately 1 h. The proteins were transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010), fixed with western closure solution (5% skim milk powder), slowly shaken on a shaker for 2 h at room temperature, and then incubated with the following primary antibodies: rabbit anti-postsynaptic density-95 (PSD-95) antibody (1:2000, abcam, ab238135), rabbit anti-synaptophysin (SYN) antibody (1:1000, Bioss, bs-8845R), rabbit anti-BDNF antibody (1:1000, abcam, ab108319), and rabbit tyrosine kinase receptor B (TrkB) antibody (1:5000, abcam, ab187041). The samples were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20000, Zsbio, ZB-2301) for 1.2 h. The membranes were washed with phosphate-buffered saline with Tween and the ECL luminescence kit (Thermo, 340,958) was used to detect the proteins. Finally, the intensity of the bands was analyzed by Image J software (Media Cybernetics, United States).