4 to 12 bis tris gel

Cells were lysed in 250 mM NaCl, 5 mM EDTA, 50 mM tris (pH 7.4), and 0.5% NP-40 containing protease inhibitors. After lysis, the lysate was spun at 15,000 rpm for 5 min. Samples boiled in SDS sample buffer containing reducing agents (Bio-Rad) were loaded and electrophoresed on a 4 to 12% bis-tris gel (Bio-Rad) for 2 hours at 120 V. The gels were transferred onto nitrocellulose membranes at 80 V for 1 hour at 4°C. The membranes were blocked in 5% bovine serum albumin (BSA) overnight at 4°C on a shaker. Primary antibodies were added in 2.5% BSA, and the membranes were incubated at room temperature for 2 hours. The membranes were washed and then incubated with rabbit or mouse secondary antibodies against horseradish peroxidase (1:5000; GE) for 1 hour at room temperature. The membranes were again washed and then developed using enhanced chemiluminescence reagent (GE).
Western blot analysis was performed using the following primary antibodies: a rabbit polyclonal antibody against Sema3D (1:1000; Abcam), a rabbit polyclonal antibody against AnxA2 (1:000; Santa Cruz Biotechnology), a rabbit polyclonal antibody against PlxnD1 (1:1000; Novus), or a mouse polyclonal antibody against β-actin (1:500; Santa Cruz Biotechnology).