Western Blot Analysis of BDNF and TrkB

BDNF and TrkB expressions were determined by Western blot analysis (Kim et al., 2006 (link); Kim et al., 2015 (link)). The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50-mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (pH, 7.5), 150-mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1-mM ethyleneglycol- bis-(b-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1.5-mM MgCl₂·6H₂O, 1-mM sodium orthovanadate, and 100-mM sodium flouride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). The protein (30 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit BDNF antibody (1:500; Santa Cruz Biotechnology), rabbit TrkB antibody (1:1,000; Santa Cruz Biotechnology), were used as the primary antibodies. Horseradish peroxidase conjugated antirabbit antibody for BDNF and TrkB (1:3,000; Vector Laboratories) and horseradish peroxidase-conjugated antimouse antibody for beta-actin (1:2,000; Vector Laboratories) were used as the secondary antibodies. Band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).