Protease and phosphatase inhibitor cocktail

Western blot analysis was conducted as previously described (Meng et al., 2014 (link)). Right cortex tissues were collected from each rat (n = 3), and the total protein was extracted using a protein extraction reagent supplemented with protease and phosphatase inhibitor cocktails (ComWin Biotech, China). The total protein concentration of each sample was determined by a BCA kit (ComWin Biotech, China). Equal amounts of protein were separated using SDS-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. These membranes were blocked before being incubated overnight at 4°C with the appropriate primary antibodies: cnpase (ab183500, 1:1000), MBP (ab209328, 1:1000), Vimentin (ab92547, 1:10,000), SYP (ab32127, 1:1000), PSD95 (ab76115, 1:1000), MAP-2 (ab32454, 1:2000), Tau-1 (ab75714, 1:1000), BDNF (ab108319, 1:1000), p-TrkB (Tyr705) (ab229908, 1:1000), TrkB (CST4603, 1:1000), p-CREB (Ser133) (ab32096, 1:1000), CREB (ab32515, 1:1000), p-Akt (Ser473) (CST4060, 1:1000), Akt (CST4685, 1:1000), and β-actin (EXP0036 F, 1:2000). Then, the membranes were washed three times and incubated with the appropriate secondary antibodies. The blots were visualized using enhanced chemiluminescence western blot detection kits (ComWin Biotech, China). The blot densities were calculated by ImageJ software.

Proteins of OCs and Tregs were harvested using RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) that was supplied with protease and phosphatase inhibitor cocktails (Beijing ComWin Biotech Co., Ltd. Beijing, China). The protein concentration was measured using bicinchoninic acid (BCA; Solarbio) protein assay. Then, the proteins were mixed with loading buffer and denatured in metal bath (100°C, 5 min). Following, the target proteins were divided via sodium dodecyl sulfate–polyacrylamide gel electrophoresis with constant voltage of 100 V and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, United States) with constant current of 300 mA in 1 h. In order to eliminate nonspecific protein binding, membranes were blocked at room temperature for 2 h with 5% skim milk (BD Biosciences) in 1 × TBST (Beyotime). And then, membranes were incubated overnight at 4°C with primary antibodies followed: phosphorylated (p-) JAK2 (1:1,000), p-STAT3 (1:2000), JAK2 (1:1,000), STAT3 (1:1,000) (Cell Signaling Technology, MA, United States). Appropriate secondary antibodies were incubated for 2 h and protein bands were detected using eECL western blot kit (Beijing ComWin Biotech Co., Ltd.). Band densities were quantified using Image Lab 3.0 software (Bio-Rad Laboratories, CA, United States).

Right cortex tissues, serum and cell lysis solution were collected from each sample, and the total protein was extracted using a protein extraction reagent supplemented with protease and phosphatase inhibitor cocktails (ComWin Biotech, Beijing, China). The total protein concentration of tissue and cell samples was determined by a BCA kit (ComWin Biotech, Beijing, China). The expression level of TNF-α, IL-1β, IL-6, MCP-1, CD11a, ICAM-1, caspase-1, NOX, PC, 4-HNE and 8-OHdG was assessed using enzyme-linked immunosorbent assay (ELISA) kits according to the previous method (Xie et al. 2019 ). All ELISA kits were purchased from Expandbio (Beijing, China).