Phospho erk

Cells and EV pellet were lysed, then stored at −80°C. Lysates (30 μg) were resolved using 10% SDS/PAGE, and proteins were transferred to nitrocellulose membrane. Membranes were incubated with primary antibody (TF: EPR8986, Abcam; phospho-ERK: E10, actin: 8H10D10, and flotillin: C42A3, Cell Signaling Technologies, Danvers, MA, USA; ERK: 16/ERK, BD Biosciences; CD9: C-4, Santa Cruz, Santa Cruz, CA, USA) overnight at 4°C, and then horseradish peroxidase-conjugated secondary antibody for 1 h. Signal was detected using chemiluminescent substrate (Thermo Scientific) and imaged by film or imager (BioSpectrum Imaging System, UVP, Upland, CA, USA). Experiments were duplicated two to three times using different lysates and EV preparations.

The total proteins of tissues and cell samples were extracted using a radioimmunoprecipitation assay buffer (Solarbio. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate–, Beijing, China)polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% bovine serum in TBST, the membranes and corresponding primary antibodies were incubated overnight in a shaker at 4°C. The primary antibodies used were as follows: anti-GAPDH (1:10,000, ProteinTech, Wuhan, China), anti-HSF2BP (1:1000, Abcam, USA), ERK (1:500, Abcam, USA), phospho-ERK (1:500, Abcam, USA), JNK (1:1,000, Abcam, USA), phospho-JNK (1:1,000, Abcam, USA), p38 (1:1,000, Abcam, USA), and phospho-p38 (1:1,000, Abcam, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:10,000, ProteinTech, Wuhan, China) at room temperature for 1 h, the membranes were visualized using the enhanced chemiluminescence method (Thermo, MA, USA) with a chemiluminescent detection system (Tanon, Shanghai, China).

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).