Imagequant tl 2005

Protein extraction and quantification were performed according to the procedures specified by the manufacturers of the reagents used. Each sample containing 20 μg of protein was separated by 5% and 10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with a primary rabbit anti-GAP-43 antibody (1:10,000), rabbit anti-CREB antibody (1:1,000), rabbit anti-p-CREB antibody (1:1,000), rabbit anti-BDNF antibody (1:20,000), rabbit anti-TrkB antibody (1:10,000), rabbit anti-PI3K antibody (1:1,000), rabbit anti-p-PI3K antibody (1:1,000), rabbit anti-Akt antibody (1:1,000), rabbit anti-p-Akt antibody (1:1,000), rabbit anti-ERK antibody (1:1,000), anti-p-ERK antibody (1:1,000), rabbit anti-PLCγ antibody (1:10,000), rabbit anti-NF200 antibody (1:1,000), rabbit anti-NF68 antibody (1:20,000), rabbit anti-NF160 antibody (1:1,000), rabbit polyclonal anti-β-tubulin antibody (1:50,000), or rabbit polyclonal anti-GAPDH antibody (1:20,000) in blocking solution at 4°C overnight. Then, the membranes were incubated with secondary goat anti-rabbit IgG (1:20,000) for 60 min and electrochemiluminescence (ECL) reagent for 30 s to 2 min followed by exposure to Kodak film (Japan). Data were represented by the IOD ratio determined using the ImageQuant TL 2005 image analysis software (Amersham, Biosciences, Piscataway, NJ).

Protein extraction (RIPA) according to the procedures specified by the manufacturers. Membranes were incubated with primarily anti-Olig1 antibody (1:2,000), anti-Olig2 antibody (1:2,000), and rabbit polyclonal anti-β-tubulin antibody (1:5,000) in blocking solution at 4 °C overnight. Data were represented by IOD ratio (ImageQuant TL 2005 image analysis software, Amersham, Biosciences, Piscataway, NJ).