Anti nt 3

For the analysis of total 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (HNE)-bound protein levels, 10 μl of hippocampus homogenate were incubated with 10 μl of Laemmli buffer containing 0.125 M Tris base pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol. The resulting samples (250 ng per well) were loaded onto a nitrocellulose membrane with a slot-blot apparatus under vacuum pressure. The membrane was blocked for 2 h with a solution of 3% (w/v) bovine serum albumin in PBS containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 and incubated respectively with primary antibodies anti-HNE (Alpha diagnostic, San Antonio, TX, USA) and anti-3NT (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at RT. Membranes were washed and incubated with anti-rabbit or mouse IgG alkaline phosphatase secondary antibodies (Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. The membrane was developed with Sigma fast tablets (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate [BCIP/NBT substrate], Sigma-Aldrich, St Louis, MO, USA). Membranes were dried and the image was acquired using ChemiDoc XP image system and analyzed using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).

Following anesthesia, the brains were removed from the skull and these were dissected into the right or left hemispheres. For protein extraction, they were placed in 10 volumes of cold homogenization buffer (120 mM NaCl, 50 mM Tris, pH 7.4) with protease inhibitors (Complete Mini, Gibco, Grand Island, NY, USA) being freshly added. Tissue then was homogenized by sonicator. Concentrations of protein were checked by the Bradford method (BioRad, Richmond, CA, USA). By adding the sampling buffer, equal amount of protein, 20 μg, was loaded and separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used with 10% polyacrylamide and 0.05% bis-acrylamide [11 (link)]. By separating proteins on the gels, they were transferred into nitrocellulose membrane. They were probed with anti-NT-3 (1 : 300, Santa Cruz, CA, USA) and anti-trkC (1 : 300, Santa Cruz, CA, USA) as primary antibody. For secondary antibody, Peroxidase anti-rabbit IgG (Vector, PI-1000, 1 : 3000 dilution) was used. As an internal control, anti-β tubulin (1 : 300, Santa Cruz, CA, USA) was checked on the sample membrane. We detected signals with enhanced chemiluminescence (Supersignal, Pierce, Rockford, IN, USA), using autoradiogram by exposing 10 to 30 min [6 (link)–8 (link)].