Different brain regions were isolated and homogenized. For injured brains, only the tissues surrounding the injury sites were collected and homogenized. Homogenates in lysis buffer (10 mM Tris-Cl, pH 7.4, 1% NP-40, 0.5% Triton-X 100, 0.2% sodium deoxycholate, 150 mM NaCl, 20% glycerol, protease inhibitor cocktail) at ratio of 1 ml per 100 mg tissue were lysed overnight in 4 °C. Lysates were centrifuged at 12 000 g and 4 °C for 30 min to get rid of the unsolved debris. Concentration of the supernatant was measured by BCA assay. Proteins in samples were separated by 6–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore), and then incubated with indicated primary antibodies at 4 °C overnight after blocking by 5% non-fat milk solution in Tris-buffered saline with Tween-20 (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Next day, the membranes were washed by Tris-buffered saline with Tween-20 for three times and incubated with the secondary antibodies for 1 h at room temperature. Membranes were washed again and incubated with substrate EZ-ECL for visualization of chemiluminescence by exposure to X-ray films or Bio-Rad GelDOCXR+ Imaging System. Intensities of protein bands were measured by Image J (NIH), and statistical analysis was performed after subtraction of the background intensity.