Ripa lysate buffer

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.).

The proteins were extracted from hippocampal tissues using RIPA lysate buffer (Beyotime, China), and then concentrations of proteins were measured using bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, China). Proteins were separated by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes, which had been washed by using Tris Buffered Saline Tween (TBST), and then were blocked with 5% skimmed milk in TBST (room temperature, 2 h), The membranes were incubated at 4°C overnight with primary antibodies. The primary antibodies included rabbit anti-NDR2 (1:3000, Affinity, USA), and rabbit anti-GAPDH (1:10,00, Servicebio, China). Unbound antibodies were washed by TBST, then the membranes were subsequently incubated with HRP-labeled goat antirabbit IgG secondary antibodies (1:200, Beyotime, China) for 1 h. The immunoreactive bands were visualized by using a chemiluminescence (ECL) kit (Beyotime, China), Images were acquired with a Chemiluminescent Gel Imaging System (FluorChem FC3, ProteinSimple, USA), and densitometric analysis was performed with ImageJ software.

Total protein of cells or tissues were extracted with RIPA lysate buffer (Beyotime, Shanghai, China). After measuring the protein concentrations by BCA protein assay, 40 μg of total protein was loaded and separated by using the 12% SDS-PAGE gels, following the transfer to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). After blocking the membrane with 5% skim milk, the membranes were incubated with the indicated primary antibodies overnight at 4 °C. Then the membranes were incubated with horseradish peroxidase-linked IgG secondary antibody (Bioworld, USA) at room temperature for 1 h. The protein band was exposed with ECL buffer following the intensity analysis with ImageJ software. The primary GPX4, Keap1, Nrf2, NOX1, COX2 antibody used in the studies was purchased from Abcam (Anti-GPX4, ab125066; Ant-Keap1, ab196346; Anti-Nrf2, ab31163; Anti-NOX1, ab55831; Anti-COX2, ab15191). ACSL4 antibody was purchased from Santa Cruz Biotechnology (Anti-ACSL4, sc-365230). FTH1 antibody was purchased from Cell signaling Technology (Anti-FTH1, #3998S). SLC7A11 polyclonal antibody was purchased from Invitrogen (Anti-SLC7A11, PA5-18599). GAPDH (ab9485, Abcam, USA) was served as a loading control.

The cell or tissue samples were lysed by a RIPA lysate buffer (Beyotime, Shanghai, China) containing 1 × protease inhibitor cocktail (Beyotime, Shanghai, China). Protein concentration was quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). The samples (20 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a PVDF membrane (BioRad, Hercules, CA, United States). Non-specific binding was blocked by incubation of the membranes in 5% defatted milk for 1 h at RT. The membranes were incubated overnight with primary antibodies at 4°C and with secondary antibody for 2 h at RT. After incubation, the membranes were washed three times with TBST. The ECL Kit (Fdbio, Zhejiang, China) was applied to the membrane, and the membrane was imaged using a ChemiDoc XRS + (BioRad, Hercules, California, United States). The gray intensity of the target band was analyzed by Image Lab software. The antibodies used are presented in Supplementary Table 2.

Furosine was purchased from PolyPeptide (Strasbourg, France); the purity was above 95%. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and non-essential amino acids (NEAA) were obtained from GIBCO (Waltham, MA, USA), 1% penicillin/streptomycin was purchased from Thermo Fisher (Waltham, MA, USA). Lactoferrin was purchased from Sigma (St. Louis, MO, USA), with the purity of above 95%.
Cell counting kit-8 (CCK-8 kit) was purchased from Solarbio (Beijing, China), Elisa detection kits of T, FSH and LH in mice serum were purchased from Jiancheng (Nanjing, China). Hematoxylin-Eosin (HE) staining kit was purchased from Solarbio (Beijing, China).
Trizol buffer, RIPA lysate buffer (including proteases and PMSF), and protein concentration detection kit were obtained from Beyotime (Nanjing, China). Primers of GAPDH, Cep55, NF-κB, PI3K, Akt, FOX01 and TNF-α were synthesized from Sangon (Shanghai, China), reagents related with quantitative real time polymerase chain reaction (q-PCR) were purchased from Sangon. Primary antibodies of β-actin, Cep55, NF-κB, PI3K, p-PI3K, Akt, p-Akt, FOX01, p-FOX01, and TNF-α, as well as the secondary antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Reagents related with western blotting were purchased from Solarbio. Enhanced chemiluminescence (ECL) reagent was purchased from Tanon (Shanghai, China).

Transfected cells were lysed with RIPA lysate buffer (Beyotime Institute of Biotechnology, Jiangsu, China) and protein lysates (40 μg) separated by using sodium dodecylsulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated overnight at 4°C with primary antibodies reactive with NLRP1 and GAPDH (1 : 1000, Abcam) and then incubated with relevant secondary antibodies (1 : 2000, Cell Signaling Technology, Beverly, MA, USA) for 1 hour at room temperature and visualized using an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, San Francisco, CA, USA). Blots were developed using a Fujifilm Las-4000 Imaging System (Fujifilm, Tokyo, Japan).