Characterizing VAV1 and CELF2 Expression in RN-Sc Cells and Spinal Cord Tissues

Western blot analysis was performed as previously described^{37 (link),38 (link)}. Proteins were extracted from RN-Sc cells and spinal cord tissues using ice-cold RIPA Lysis Buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Thermo Scientific) and quantified using the Bio-Rad Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Next, proteins (30 μg/sample) were subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocked with 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies against VAV1 (#2502, 1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA), CELF2 (ab186430, 1:2000 dilution, Abcam, Cambridge, UK), and β-actin (ab6276, 1:5000 dilution, Abcam). Next, the membranes were incubated for 1 h at room temperature with goat-anti-rabbit/mouse secondary antibody conjugated with horseradish peroxidase (ab205718/ab205719, 1:5000 dilution, Abcam). Finally, proteins bands were visualized through SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).