Bca analysis tool kit

The cells and spinal cord tissue (1 cm long) were lysed with RIPA containing 1% PMSF, and the protein was collected after ultrasound treatment and ultracentrifugation at 12,000 rpm for 30 min. After detecting the protein concentration with BCA analysis tool kit (Beyotime, Shanghai, China), the protein concentration was fixed to a specific level (80 μg in vivo and 40 μg in vitro). The proteins were separated on 10–12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20), the membranes were incubated overnight at 4 °C with primary antibodies. The membranes were washed thrice with TBST for 3 min each time and incubated with HRP-conjugated IgG secondary antibody at room temperature for 1 h. Finally, after washing three times with TBST, the membrane blot was detected using the Chemi DocXRS + imaging system (Bio-Rad, USA) with a super-sensitive ECL chemiluminescence kit (Epizyme Biomedical Technology, Shanghai, China), and quantitative analysis was performed using the Image J software [62 ]. The concentration of antibody used in western blot is shown in Supplementary Table 2.

Total protein was collected from cultivated NP cells or milled tissues using ice-cold RIPA lysis buffer (Beyotime) involving 1% phenylmethanesulfonyl fluoride (PMSF). The protein concentration of cell lysates was determined using a BCA analysis tool Kit (Beyotime). Next, the protein of NP cells for each sample was separated via gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). After blocking using 5% nonfat milk diluted in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at room temperature, membranes were incubated with the primary antibodies overnight at 4 °C. Following washing three times with TBST, the membranes were incubated with respective secondary antibodies for 2 h at room temperature. Finally, the blots were detected with electrochemiluminescence reagent, and the densitometry of these blots was quantified using Image Lab V3.0 software (Bio-Rad).