Sciatic Nerve Protein Analysis

Proteins expressed in the sciatic nerve were determined by western blot analysis. Sciatic nerves were rapidly dissected from rats and used for protein analysis. All the procedures including protein extraction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and western blotting were essentially the same as described in our previous report [8 (link)]. Briefly, cells in the sciatic nerves were lysed using triton lysis buffer, sonicated, and centrifuged to collect the supernatant containing proteins. Protein (15 μg) was used for SDS-PAGE (10%) and western blotting analyses. Anti-phospho-Erk1/2 (1 : 1250, rabbit polyclonal, Cell Signaling, Seattle, USA), anti-p38 (1 : 1250, rabbit polyclonal, Santa Cruz Biotech.), anti-TNF-α (1 : 1250, rabbit polyclonal, Sigma), and anti-actin (1 : 10,000, mouse monoclonal, MP Biomedicals, Santa Ana, USA) antibodies were used as primary antibodies, and horseradish peroxidase- (HRP-) conjugated antibody (1 : 1250; goat anti-rabbit or goat anti-mouse, Santa Cruz Biotech.) as a secondary antibody. Protein band intensity in the scanned images of X-ray film was determined by using the i-Solution software (Image & Microscope Technology, Daejeon, Korea).