Anti asc

Protein from tissues of spinal dorsal horn was extracted in RIPA lysis buffer, which was bought from Beyotime Biotechnology of China. Through BCA Protein Assay (Thermo Fisher Scientific), the protein concentration was assessed. To separate 30 μg of protein samples, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then utilized. After separation, protein samples were placed in a polyvinylidene difluoride (PVDF) nitrocellulose membrane (Millipore, Boston, MA, United States), arrested by 5% fat-free dry milk at 25°C in TBST for 1 h and cultured at 4°C with primary antibodies overnight. Primary antibodies included the following: anti-ASC (1:500; ABclonal), anti-Nlrp3 (1:1000; Proteintech), anti-GSDMD (1:1000; Abcam), anti-cleaved Caspase-1 (1:1000; Proteintech), anti- NF-κB/p65 (1:2000; Abcam), and anti-GAPDH (1:10,000; Cell Signaling Technology, CST). The membranes were cultured with a rabbit or mouse HRP-conjugated secondary antibody (1:2000; Beyotime Biotechnology) at 25°C for 1 h after being washed in TBST. Finally, the densities of the target proteins relative to that of GAPDH were quantified using Image Pro Plus software.

Lysis and protein extraction of spinal cord tissues or astrocytes was performed using the RIPA lysate buffer (P0013; Beyotime). The concentration of the extracted protein was determined by the BCA Protein Assay Kit (P0011; Beyotime). The protein (20–40 µg per lane) was separated on 8–15% SDS-PAGE gel and transferred to polyvinylidene fluoride membrane (IPVH00010; Millipore, Billerica, MA, USA). After blocking with 5% (m/v) skim milk for 1 h at room temperature, the membrane was incubated at 4 °C overnight with primary antibodies in skim milk at a dilution of 1:1000. These primary antibodies were as follows: anti-HSPA8 (A14001; Abclonal), anti-NLRP3 (A5652; Abclonal), anti-ASC (A1170; Abclonal), anti-caspase-1 (A0964; Abclonal), anti-NF-κB P65 (AF5006; Affinity, Cincinnati, OH, USA), anti-phosphorylated NF-κB P65 (p-NF-κB P65) (AF2006; Affinity), anti-IL-1β (A16288; Abclonal), anti-IL-18 (A16737; Abclonal), and anti-β-actin (sc-47778; Santa Cruz). After washing with Tris-buffered saline-Tween 20 (TBST) buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:3000 dilution; A0208; Beyotime) or mouse (1:3000 dilution; A0216; Beyotime) secondary antibody at 37 °C for 40 min. The membranes were visualized using a chemiluminescence kit (Shanghai 7sea biotech Co. Ltd.) and analyzed using Gel-Pro-Analyzer 4.0 (Media Cybernetics, Inc.).

Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory. BSA and rhTrx‐1 were purchased from R&D System. Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio. ROS and Necrostatin‐1 was purchased from Sigma. Annexin V‐PE/7AAD assay was purchased from BD Biosciences. All the ELISA kits were purchased from Cusabio. Dapi dyeing solution and JC‐1 kit were purchased from Beyotime. DMEM, foetal bovine serum (FBS) and MEM/EBSS were purchased from Hyclone. The primary antibodies used for immunofluorescence staining: Rabbit polyclonal anti‐Iba‐1 (Wako), Goat polyclonal anti‐Iba‐1 (Abcam), Mouse monoclonal anti‐RIPK1 (Santa cruz), Goat polyclonal anti‐CD206 (R&D System) and Rabbit polyclonal anti‐CD16 (Bioss). All the second antibodies used for immunofluorescence staining were purchased from Abcam company. The primary antibodies used for Western blot analysis: anti‐RIPK1, anti‐RIPK3, anti‐MLKL, anti‐pMLKL and anti‐CCL2 (Abcam); anti‐NLRP3, anti‐ASC, anti‐caspase‐1, anti‐caspase‐3 and anti‐β‐actin (ABclonal); anti‐MMP‐9 (R&D System); and anti‐CD16 (Bioss). The second antibodies used or Western blot analysis was Alexa Fluor 800‐conjugated Goat‐anti rabbit (LI‐COR).