Anti p p38

28 days following SCI, euthanized rats under isoflurane anesthesia. Samples were then collected and fixed in 4% paraformaldehyde. A 0.5 cm long sample tissue was homogenized in 300 μL of lysate-containing protease inhibitors. Following lysate preparation, the electrophoresed tissue lysate was transferred to polyvinylidene difluoride membranes. Next, the membranes were blocked with 5% skimmed milk and incubated in an incubator for 60 min. The membranes were further blocked for two hours and then incubated with primary antibodies, consisting of anti-JNK (1:1000; Proteintech), anti-p-JNK (1:1000; Proteintech), anti-p-p38 (1:500; Bioss), anti-p38 (1:1000; Bioss), and GAPDH (1:2000, UtiBody). They were then incubated with secondary antibodies (1:3000; Bioss) in an incubator for 1 h. ImageJ was used to quantify grey bands.

IL-1β was obtained from T&L Biotechnology Co., LTD. (Beijing, China). DMEM medium and FBS were obtained from Genetime Biotechnology Co., Ltd. (Shanghai, China). Primescript™ reverse transcription kit and SYBR^® Premix Ex Taq™ II were purchased from TransGen Biotech (Beijing, China). Antibodies including anti-MMP-1, anti-MMP-3, anti-MMP-9, anti-ADAMS-4, anti-ADAMS-5, anti-collagen, anti-aggrecan, anti-iNOS, anti-COX-2, anti-p-p38, anti-p-ERK1/2, and anti-p-p65 were all obtained from Bioss Biotechnology Co., Ltd. (Beijing, China). GAPDH was purchased from Abcam Inc. (Cambridge, U.K.). The sequences of primers used in the present study was obtained from Tsingke (Beijing, China). ELISA kits were purchased from MSKBIO Technology Ltd. (Wuhan, China).

D-gal (≥99%) was purchased from Sigma Aldrich (St. Louis, MO, USA). Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and 8-hydroxy deoxyguanosine (8-OHdG) commercial kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Testosterone, cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-β1 (TNF-β1), and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Nanjing SenBeiJia Biological Technology Co., Ltd (Nanjing, China). Anti-p-p38, anti-p-ERK1/2, anti-p-JNK, anti-p38, anti-ERK1/2 and anti-JNK antibodies were purchased from Bioss (Beijing, China). The primary antibody for GAPDH was from Biogot Technology Co., Ltd. (Nanjing, Jiangsu, China). Anti-rabbit secondary antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). All other reagents used in the experiments were of analytical grade.