Western Blotting

OPCs were plated at a density 2.7 × 10⁵ cells/ml and were treated with 10 ng/ml of recombinant mouse leptin. After culturing, the cells were collected in lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Triton-X 100, and 50 μM EDTA) containing a cocktail protease inhibitors (Roche). The lysates were clarified by centrifugation at 15,900 × g at 4 °C for 10 min, and the supernatants were collected and normalized for protein concentration. Proteins were separated by 9% SDS-PAGE, and then transferred onto polyvinylidene fluoride membranes (Immobilon-P, Millipore). The membranes were incubated with PBS containing 5% skim milk and 0.05% Tween 20 for 1 h at room temperature following incubation with primary antibodies. Antibody binding was detected by an electro-generated chemiluminescence system (Amersham Imager 600, GE Healthcare). The intensity of protein bands was quantified using ImageJ software (NIH). The following antibodies were used: anti-phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, 9101 S, Cell Signaling Technology); anti-p44/42 MAPK (ERK 1/2) (1:1000, 9102 S, Cell Signaling Technology); and horseradish peroxide (HRP-conjugated anti-rabbit IgG (1:5000, eB182, TrueBlot).