Western Blot Analysis of Neuronal Proteins

Western blot assays were followed according to an established protocol^{32 (link)}. Briefly, dissected cerebral cortex tissues, cervical spinal cord tissues or cells were lysed in RIPA lysis buffer (Cell Signal Technology, Beverly, MA). Protein concentrations were determined using a BCA assay (Pierce, Roclford, IL, USA) and equal amounts were loaded onto 10% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and separated by SDS-PAGE. Samples were then transferred to PVDF membranes, blocked in 5% nonfat dried milk (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and incubated overnight at 4 °C with primary antibodies. Antibody labelling was detected by incubation with horseradish peroxidase-labelled anti-rabbit/mouse secondary antibody (Proteintech, Wuhan, China). Protein was visualized using chemiluminescence (Millipore). Antibodies evaluated by Western blot included rabbit anti-p-PKCγ (Abcam), rabbit anti-GAP43 (Abcam), mouse anti-β-Catenin (Abmart), rabbit anti-phospho-GSK-3β (Ser9) (Cell Signal Technology), mouse anti-GAPDH (Thermo Fisher Scientific), mouse anti-β-actin (Thermo Fisher Scientific), mouse anti-HA-tag (Abmart), and mouse anti-GFP (Abcam).