Myelin basic protein

Cells grown in 60 mm dishes (3.0×10⁵ cells/ml) were rinsed with PBS and then centrifuged at 12,000 g for 30 min at 4°C. After suspension in RIPA lysis buffer (Millipore, Billerica, MA, USA) for 30 min on ice, protein concentrations were determined by Bradford assay. Following the addition of loading buffer and boiling for 5 min, equal amounts of protein from each sample were subjected to SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were first blocked with 1% non-fat dried milk in 10 mM PBS with 0.05% Tween-20 (PBST) for 2 h at room temperature and then incubated overnight at 4°C with antibodies targeting the following proteins: CNPase (1:500, Boster), myelin basic protein (MBP) (1:500, Santa Cruz Biotechnology, CA, USA), phosphorylated ERK1/2 (p-ERK1/2) (1:500, Cell Signaling Technology, Beverly, MA, USA), ERK1/2 (1:500, Cell Signaling), activated poly-ADP-ribose polymerase-1 (PARP-1) (1:500, Santa Cruz), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000, Bioworld Technology, Inc., USA). After washing with PBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz) at 37°C for 3 h. Protein bands were detected by the enhanced chemiluminescence method (ECL kit, Amersham, UK).