Western blot analysis for the NF-200 and BDNF was performed as the previously described method (Jung et al., 2016 (link); Kim et al., 2017 (link); Park et al., 2017 (link)). The sciatic nerve tissues were collected, and then were immediately frozen at −70°C. After homogenizing sciatic nerve tissues, tissue were lysed in a lysis buffer containing 50 mM HEPES (pH, 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl2·6H2O, 1 mM sodium orthovanadate, and 100 mM sodium flouride. Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to determine protein content. Protein (30 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane.
For the primary antibody, mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology), mouse NF-200 antibody (1:1,000; Santa Cruz Biotechnology), and rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology) were used. As the secondary antibody, horseradish peroxidase-conjugated anti-mouse antibody (1:3,000; Amersham Pharmacia Biotechnology GmbH, Freiburg, Germany) for beat-actin and NF-200, and anti-rabbit antibody (1:2,000; Vector Laboratories) for BDNF were used. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for the band detection.