L1cam

Cultured cells were solubilized in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis sample buffer, and the protein concentration in each sample was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. Proteins (30 µg of protein per lane) were then resolved in a 7.5%–10% SDS polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane. Membranes were blocked with 4% nonfat milk in TBST (Tris-HCl based buffer with 0.2% Tween 20, pH 7.5) and then incubated in the presence of primary antibody overnight at 4 °C. Cells were then incubated for 1 hour in the presence of a 1:5,000 dilution of secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratory, West Grove, PA). Reaction product was visualized using an enhanced chemiluminescence Western blot detection kit (Amersham Bioscience, Piscataway, NJ). The primary antibodies used were: a mouse monoclonal antibody against TRF2 (Imgenex); a goat polyclonal antibody against REST (P18, Santa Cruz); and antibodies against Olig2 (Millipore), p53 (Cell Signaling), β3-tubulin (Sigma), L1CAM and actin (Sigma).