*5.4. Biochemical Studies*

#### Western Blot Analysis

Spinal cord tissues (L4–L6) were collected and homogenized in a lysis buffer containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentrations of the samples were determined using a Bradford assay [36]. Total protein of 30 μg was separated on SDS-PAGE gel (10% gradient gel) and transferred to nitrocellulose membranes (BioRad, Santo Amaro, SP, Brazil). The membranes were blocked for 120 min with 5% bovine serum albumin (BSA) or non-fatty milk and incubated overnight at 4 ◦C with a primary antibody against phospho-ERK, phospho-JNK, phospho-p38 or non-phosphorylated forms of these proteins (1:1000; Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated in the correspondent peroxidase-conjugated secondary antibody (1:5000; antirabbit or antimouse, Sigma-Aldrich, St. Louis, MO, USA, cat. numbers A0545 and A8919, respectively) for 120 min at room temperature. The enhanced chemiluminescence method was used to develop the filters (Amersham GE Healthcare Bio-Sciences Corp.; Piscataway, NJ, USA). The densitometric data were analyzed using the UVITEC Cambridge software (UVITEC Cambridge, Cambridge, UK) and normalized to the total protein (not phosphorylated).
