Mice were first euthanized by carbon dioxide asphyxiation and spinal cord tissue was harvested by extrusion. Lumbar spinal cord segments were dissected on a chilled surface. The lumbar area of the spinal cord was readily identified by its enlargement, and the area spanning the approximate L3 through S1 segments was harvested. Dissected tissue was then quick-frozen in liquid nitrogen and stored at -80°C until required for analysis. Spinal cords were dissolved at 4°C in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) in the presence of protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein (20 μg) were loaded for SDS-PAGE (10% Tris–HCl acrylamide gel) and electrotransferred onto a polyvinylidene difluorided membranes as described previously [54]. Blots were probed with the primary antibody of DCC monocolonal antibody (1:1000, BD Pharmingen, San Diego, CA) at 4°C overnight. β-actin (Sigma Chemical, St. Louis, MO) was used as an internal control. This antibody produces a single band at approximately ~185 kDa on Western blots consistent with the predicted weight
