Ripa solution

The cells or tissues were lysed in RIPA solution (P0013B, Beyotime, Haimen, China) and quantified using BCA protein assay (P0012, Beyotime). The protein (50 µg) was separated on SDS-PAGE and then transferred to PVDF membranes (3010040001, Roche, Basel, Switzerland). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: PDGFR-α (ab203491, 1:1000, Abcam), PDGFR-β (ab69506, 1:1000, Abcam), α-SMA (ab124964, 1:2000, Abcam), NG-2 (ab275024, 1:1000, Abcam), collagen I (ab260043, 1:1000, Abcam), desmin (ab227651, 1:5000, Abcam), KDM5B (ab181089, 1:1000, Abcam), H3K4me2 (#9725, 1:1000, CST, USA), H3K4me3 (#9751, 1:1000, CST, Danvers, MA, USA), and β-actin (ab8226, 1:1000, Abcam). After incubation with a secondary antibody, the membranes were treated with the ECL-chemiluminescent kit (34580, Thermo Fisher Scientific) to visualize the protein bands.

One centimetre of spinal cord tissue containing the central area of SCI was taken for protein extraction. Briefly, spinal cord tissue was homogenized in 1 mL of RIPA solution (Beyotime, China) and centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected for protein quantification. Twenty micrograms of protein was loaded onto a sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore, USA). After the membrane was blocked at room temperature for 30 min, EphA4, ephrin-B1, ephrin-B2 and ephrin-B3 primary antibodies (Abcam, USA) were separately incubated overnight at 4 °C, goat anti-mouse HRP (CST, USA) or goat anti-rabbit HRP (CST, USA) secondary antibodies were used in combination with primary antibodies, and an ECL Plus kit was used to detect the positive bands with an Odyssey dual colour infrared laser imaging system (Odyssey, USA). GAPDH was used as the internal reference. Protein quantification was performed using IPP software, and the differences between groups were determined.

The protein expression of N-CDH was analysed by Western blotting. Briefly, the total protein was extracted from isolated porcine disc NP tissue using RIPA solution (Beyotime, China). The protein samples were subjected to SDS/PAGE electrophoresis and transferred on to PVDF membranes. After membranes were incubated with primary antibodies (N-CDH, Abcam, ab18203, diluted 1:1000; β-actin, Proteintech, 60008-1-Ig, diluted 1:2000) at 4°C overnight and the corresponding secondary antibodies (ZSGB-BIO, China, diluted 1:2000) at 37°C for 2 h, the protein bands were developed using a SuperSignal West Pico Trial Kit (Thermo, U.S.A.). After the grey values of protein bands were analysed using ImageJ software (National Institutes of Health, U.S.A.), the protein expression of N-CDH was normalized to that of β-actin. In addition, the protein bands transferred on to the PVDF membrane were visualized by Ponceau staining.

OCs were lysed in RIPA solution (Beyotime) to extract total proteins, which were quantified as per instructions from a BCA kit (Thermo Fisher Scientific). Protein samples (20 μg) were loaded onto 8% to 15% SDS-PAGE gel, separated, and transferred to PVDF membrane (Millipore, Shanghai). After blocking with 5% skim milk, primary antibodies against Abca1 (1:1,000, affinity, Liyang, China), Bax (1:1,000, affinity), Bcl-2 (1:1,000, affinity), beta-actin (1:3,000, Bioss, Beijing, China), Caspase-3 (1:1,000, Cell Signaling Technology), c-Fos (1:1,000, Abcam, Cambridge, UK), COX IV (1:1,000, Abbkine, Wuhan, China), CTSK (1:1,000, Abcam), Cytochrome c (1:1,000, affinity), ERK (1:1,000, Abbkine), IκB-α (1:1,000, Signalwayκ Antibody, Maryland, USA), MMP9 (1:500, ProteinTech, Wuhan, China), NFATc1 (1:1,000, Cell Signaling Technology, Danvers, USA), p-ERK (1:1,000, ProteinTech), and tubulin (1:1,000, affinity) were used to detect the target proteins. After washing for 30 min (5 min * 6 times) with TBST, corresponding secondary antibodies purchased from Abbkine (1:8000) were incubated for 2 h at RT. The images were taken through a Bio-Rad imager after developing the membrane with a HRP substrate. Band intensities were analyzed by the Image J software.

Protein was extracted from HUVECs by RIPA solution (P0013B, Beyotime) combined with phenylmethanesulfonyl fluoride (PMSF) and a phosphatase inhibitor. Then, all the protein samples were mixed with 1X SDS–PAGE protein loading buffer (P0015A, Beyotime). After all the samples were denatured at 100°C for 10 min, electrophoresis was performed using a 10% SDS–PAGE gel preparation (Bio-Rad, USA) at 80 V for 30 min and 120 V for 90 min. Then, the proteins were blotted onto a di-fluoride polyvinylidene fluoride (PVDF) membrane at 240 mA for 2 h in ice water. Next, the PVDF membrane was blocked in QuickBlock™ buffer (P0252, Beyotime) for 40 min and then incubated with diluted primary antibodies (Table 3) at 4°C overnight. After washing three times with tris buffered saline with tween (TBST), horseradish-peroxidase-conjugated (HRP-conjugated) antibodies were added to the PVDF membrane. After three washes, the immunoreactive bands were visualized using ChemiDoc Touch (Bio-Rad, USA). We used a Spectra multicolour high-range protein ladder (26 625, Thermo Scientific) as the protein ladder.

Briefly, after the total protein was extracted using RIPA solution (Beyotime, China) with frequent agitation for 20 min and protein concentration was measured using a BCA chemical kit (Beyotime, China), equal amount of protein samples in each group was separated using SDS/PAGE and transferred on to the PVDF membrane. Then, the PVDF membrane was incubated overnight at 4°C with the primary antibodies (N-CDH, Abcam, ab18203, diluted 1:1000; β-actin, Proteintech, 60008-1-Ig, diluted 1:2000),and corresponding HRP-conjugated secondary antibodies (goat antimouse IgG and goat antirabbit IgG, ZSGB-BIO, China, diluted 1:2000) for 2 h at 37°C. Protein bands in the PVDF membrane were developed using a SuperSignal West Pico Trial Kit (Thermo, U.S.A.). The gray value of protein bands was analyzed using the ImageJ software (National Institutes of Health, U.S.A.). Protein expression of the target molecule was expressed as the ratio of the gray value of target molecule to that of β-actin.