Phospho trkb

PSP contained 72.12% carbohydrate (Mw = 20.48 KDa) which was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Infrared spectroscopy showed that the PSP samples had typical polysaccharide structure characteristics.
The BV2 mouse microglial line was purchased from Wuhan Sunncell Bio-Technology Co., Ltd. (Wuhan, China); fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) from Gibco (Grand Island, NE, USA); lipopolysaccharides (LPS) from Sigma (St. Louis, MO, USA); enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor (TNF-α), interleukin (IL)-6, IL-1β, and IL-10 from Jianglai Biological Technology Co., Ltd. (Shanghai, China); and the Nitric Oxide (NO) assay kit, CCK-8 assay kit, and DCFH-DA Reactive Oxygen (ROS) Fluorescent Probe from Solarbio Science and Technology Co., Ltd. (Beijing, China). Antibodies targeting iNOS, BDNF, ERK, Phospho-ERK, CREB, Phospho-CREB, TrkB, and Phospho-TrkB from Abcam (Cambridge, UK); antibody targeting Arginase-1 from Cell Signaling Technology (Boston, MA, USA); antibodies targeting Iba-1, Notch1, and Hes1 from Santacruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit AlexFluor 488^® secondary antibodies from Servicebio Co., Ltd. (Shanghai, China); and CD16/CD32 monoclonal antibody PE and CD206 (MMR) monoclonal antibody PE from Ebioscience (San Diego, CA, USA).

After homogenization in RIPA lysis buffer (with protease/phosphatase inhibitors), the hippocampal homogenate was centrifuged (4°C; 12,000 g; 15 min), and quantified via the BCA protein assay. Next, the samples (30 μg) were electrophoresed (10% SDS-PAGE), and then transferred onto a PVDF membrane (Millipore, USA). The non-specific sites on the membrane were blocked by incubation for 1 h in 5% non-fat dry milk in TBS-T. Next, the membranes were kept in overnight incubation at 4°C with primary antibodies, including mTOR (1:1000, Cell Signaling); phospho-Akt (1:2000, Cell Signaling); phospho-TrkB (1:1000, Abcam); phospho-mTOR (1:1000, Cell Signaling); BDNF (1:5000, Abcam), and β-actin (1:1000, Cell Signaling). After washing, the membrane was incubated for 1 h with HRP-conjugated secondary antibody at room temperature. The enhanced chemiluminescence method was used to visualize the bands, which were captured using ChemiDoc XRS (Bio-Rad, USA). To eliminate variations in protein expression, three independent experiments were performed, and the data were adjusted to correspond to internal reference expression (β-actin), and the results are shown as a percentage of controls.