Bicinchoninic acid kit

Total protein was extracted from cells and tissues with SDS lysis buffer (Beyotime), with the concentration measured utilizing a bicinchoninic acid kit (20201ES76, YEASEN Biotechnology Co., Ltd., Shanghai, China). After separation using 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the sample was submitted to electrotransfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) which were blocked using 5% blocking solution with skimmed milk powder and underwent overnight incubation at 4°C with primary rabbit antibodies against CGRP (ab189786, 1 : 1000, Abcam), RUNX2 (ab76956, 1 : 1000, Abcam), OCN (ab93876, 1 : 500, Abcam), ALP (ab229126, 1 : 1000, Abcam), CD45 (ab40763, 1 : 5000, Abcam), CD73 (ab133582, 1 : 5000, Abcam), and CD90 (ab92574, 1 : 1000, Abcam) as well as 1 h-incubation with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (ab150077, 1: 1000, Abcam) at ambient temperature. The immunocomplexes on the membrane were visualized utilizing enhanced chemiluminescence (ECL) reagent (ECL808-25, Biomiga, USA) at ambient temperature for 1 min, and band intensities were quantified with the help of Image Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD, USA).

Radio immunoprecipitation assay lysis buffer (Yeasen, China) was applied to extract proteins from PC-12 cells or spinal cord tissues. Protein concentration was determined with a bicinchoninic acid kit (Yeasen, China). Equal amounts of protein (30 μg) in all samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride films (Yeasen, China). Nonspecific protein binding was blocked using 5% nonfat milk in 0.1% Tris Buffered Saline Tween for 2 h at room temperature. Next, the membranes were incubated with primary antibodies against interleukin (IL)-6 (1:1000, Abcam), IL-1β (1:1000, Abcam), tumor necrosis factor alpha (TNF-α; 1:1000, Abcam), B-cell lymphoma-2 (Bcl-2; 1:1000, CST), Bcl-2 associated X (Bax; 1:1000, CST), pro caspase-3 (1:1000, Abcam), cleaved caspase-3 (1:500, Abcam), NOX4 (1:2000, Abcam) and GAPDH (1:1000, CST) overnight at 4°C. Next, the horseradish peroxidase-marked secondary antibody was added and incubated for 1 h. The bands were visualized with the enhanced chemiluminescent kit (Yeasen, China). The expression levels of proteins were analyzed by the Image J software (version ImageJ 1.44P; National Institute of Health) to determine gray density.

Tissue or cell total protein was extracted using Radio Immunoprecipitation Assay lysis buffer (R0010, Solarbio, Beijing, China) at 4 °C for 15 min followed by centrifugation at 15,000 r/min for 15 min. The protein concentration was measured using a bicinchoninic acid kit (20201ES76, Yeasen, Shanghai, China). Then, the proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto the PVDF membrane, and blocked in 5% bovine serum albumin for 1 h. Next, the membrane was incubated at 4 °C overnight with diluted primary antibodies: Fbxw7 (ab109617, 1: 200), EZH2 (ab186006, 1: 500), ZBTB16 (ab39354, 1: 1000), FLAG (ab1162, 1: 200), HA (ab9110, 1:2000), Myc (ab9106, 1:500), GAPDH (ab8245, 1:1000), and Ub (ab7780, 1:500). All the rabbit polyclonal antibodies were purchased from Abcam (Cambridge, UK) unless otherwise noted. After incubation, the membrane was re-probed with horseradish peroxidase-labeled anti-Rabbit immunoglobulin G (IgG) H&L (ab205718, 1:10,000) for 1 h at room temperature. The blots were visualized with enhanced chemiluminescence regents. The images were captured using FUSION FX5 (Vilber Lourmar, France), and analyzed by ImageJ 1.48u (National Institutes of Health, USA). GAPDH (ab9485, 1:500) was served as the internal reference.

The total protein of tissues and cells was extracted by high-efficiency RIPA lysis buffer (R0010, Solarbio). After centrifugation at 15,000 rpm/min for 15 min and lysis at 4°C for 15 min, the supernatant was harvested, and the protein concentration was evaluated employing the bicinchoninic acid kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Following separation utilizing polyacrylamide gel electrophoresis, the protein was transferred onto PVDF membranes which were sealed at 5% BSA for 1 h at ambient temperature and probed with the diluted primary antirabbit antibodies (all from Abcam) against Sesn2 (ab178518, 1: 10000), Srx1 (ab203613, 1: 10000), Trx1 (ab273877, 1: 10000), Beclin-1 (ab210498, 1: 10000), LC3A/B (ab128025, 1: 1000), Ki67 (ab92742, 1: 10000), Aggrecan (ab3778, 1: 10000), MMP3 (ab39012, 1: 10000), GRP94 (ab238126, 1: 10000), APOB (ab20737, 1: 10000), and GAPDH (ab8245, 1: 10000) overnight at 4°C. The next day, the membrane was reprobed with HRP-labeled goat antirabbit IgG (ab205718, 1: 20000, Abcam) for 1 h at ambient temperature. The developing solution was added for development. ImageJ 1.48 software (National Institutes of Health) was employed for protein quantitative analysis, and protein quantitative analysis was implemented with the gray value ratio of each protein to the internal reference GAPDH.

The total protein of cells and tissues were extracted using high efficiency RIPA lysis buffer (R0010, Beijing Solarbio Science and Technology Co. Ltd., Beijing, China). The samples were centrifuged at 15,000 rpm/min for 1 min. Then, the supernatant was collected to measure the protein concentration using a bicinchoninic acid kit (20201ES76, Yeasen Company, Shanghai, China). The protein was separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane using wet transfer method. The membrane was blocked with 5% BSA at room temperature for 1 h, and incubated with primary rabbit polyclonal antibodies to NLRX1 (ab105412, 1: 1,000), Bcl-2-Associated X (Bax; ab53154, 1: 1,000), B-cell lymphoma-2 (Bcl-2; ab196495, 1: 1,000), Cleaved-caspase-3 (ab2302, 1: 500) and GAPDH (ab181602, 1: 10,000) at 4° C overnight. Afterwards, the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit antibody to IgG (ab205718, 1: 20,000) at room temperature for 1 h. All above antibodies were purchased from Abcam Inc. (Cambridge, UK). Signals were detected by chemiluminescence and densitometry was performed utilizing ImageJ 1.48u software (National Institutes of Health, Bethesda, Maryland, USA). The relative expression was calculated with the ratio of gray value of protein bands to that of GAPDH.