Radioimmunoprecipitation assay lysis buffer

Total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (Boster, Wuhan, China). Protein concentrations were determined using the bicinchoninic acid assay (Boster, Wuhan, China). Protein (30 μg) was separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and later electroblotted onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% skimmed milk at 37 °C for 2 h and incubated overnight at 4 ℃with CUL2 antibody (Invitrogen, CA, USA) or β-actin antibody (CST, MA, USA). The following day, the membranes were incubated with anti-HRP rabbit antibody at 37 ℃ for 1 h and treated with an ultra-high sensitivity enhanced chemiluminescence reagent (Boster, Wuhan, China).

Total proteins were extracted from cells using a radioimmunoprecipitation assay lysis buffer (Boster, Wuhan, China) according to the manufacturer’s instructions. Then, the proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Lahiri et al., 2020 (link)) and transferred to polyvinylidene difluoride membranes (Millipore Corporation) (Mossoba et al., 2020 (link); Yang et al., 2020 (link)). After blocking with 5% skimmed milk in Tris-buffered saline/Tween-20 for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: Bcl-2 (1:1,000; #3498, Cell Signaling Technology, Danvers, MA, United States), Bax (1:1,000; #2772, Cell Signaling Technology), caspase-9 (1:1,000; #9504, Cell Signaling Technology), c-IAP (1:1,000; #4952, Cell Signaling Technology), survivin (1:1,000; #2808, Cell Signaling Technology), and Bad (1:1,000; #ab90435, Abcam, Cambridge, United Kingdom). The membranes were washed three times with Tris-buffered saline/Tween-20 and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:3,000; Abcam) for 1 h at room temperature. Images were visualized using a chemiluminescent substrate (Boster) and analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, United States) (García-Gómez and Valiente, 2020 (link)).

Cells were collected by detachment with trypsin and then lysed in radio immunoprecipitation assay lysis buffer (Boster, Hubei, China), followed by estimation of protein concentration using a bicinchoninic acid quantification kit (Boster). Subsequent to separation using freshly-prepared 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the protein was electro-transferred onto polyvinylidene fluoride membranes. The membrane underwent 5% bovine serum albumin (BSA) blocking and probing with primary antibodies (Abcam, Cambridge, UK) to KDM2A (ab191387, 1:1000), JAG1 (ab7771, 1:500) and GAPDH (ab181602, 1:500). Following incubation with secondary antibody goat anti-rabbit immunoglobulin G (IgG) (ab205719, 1:500, Abcam), immunoblots were visualized with enhanced chemiluminescence detection reagents (Millipore Corp., Bedford, MA, USA) and then captured under the Bio-Rad image system (Bio-Rad, Hercules, CA, USA). Gray values of target protein bands were quantified using Image J software, with GAPDH as a normalizer.

Tissues from the gray matter at the spinal lesion sites were collected and lysed in radio-immunoprecipitation assay lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), and the protein concentration was determined using a bicinchoninic acid kit (70-PQ0012, MultiSciences, Zhejiang, China). Then, the protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA for 1 h and then incubated with the primary antibodies to RGMA (1:10000, ab169761, Abcam), NF-200 (1:1000, #55453, CST), synaptophysin (1:1000, ab14692, Abcam), STAT3 (1:1000, ab68153, Abcam), p-STAT3 (1:1000, ab32143, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000, ab9485, Abcam) at 4 °C overnight. Next, the membranes were incubated with the horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:2000, ab205719, Abcam) at room temperature for 1 h. The western blots were developed using an enhanced chemiluminescence kit (BB-3501, Amersham Pharmacia Biotech, Piscataway, NJ, USA) exposed by a Gel imager, captured by a Bio-Rad image analysis system (Bio-Rad, Inc., Hercules, CA, USA), and analyzed using the Quantity One v4.6.2 software.