Rabbit anti p p38 mapk

Three hours after the injection of zymosan, three rats from each of the control, zymosan-vehicle, and zymosan-BD1047 (100 mg/kg) groups had the right side of their L_4-5 spinal dorsal horn (lamina I-V) or L_4-5 DRG removed under anesthesia with 5% isoflurane. The spinal cord or DRG was homogenized in buffer containing 1 M Tris (pH 7.5), 1% NP-40, 0.5 M EDTA (pH 7.5), 50 mM EGTA, 1 M dithiothreitol, 1 M benzanidine and 0.1 M PMSF. The total amount of protein in each sample was determined using a Bradford dye assay before each was loaded onto polyacrylamide gels. The tissue homogenates (30 µg protein) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. After washing the blots with TBST (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), the membranes were blocked with 5% skim milk for 1 h. The samples were incubated with primary antibodies for rabbit anti-CCL2 (Millipore, 1:1000), rabbit-Iba1 (Wako, VA, USA, 1:1000), and rabbit anti-p-p38 MAPK (Cell Signaling, 1:500). β-actin antibody was used as a loading control (Sigma). After the secondary antibody reaction, the bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Bio-tech, Bukinghamshire, UK).