Molecular Profiling of Hippocampal Signaling

Western blot for the expressions of Bax, Bcl-2, BDNF, TrkB, CREB, and p-CREB were performed, according to the previously described method (Kim et al., 2014 (link); Ko et al., 2009 (link)). The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH, 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membranes were incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated overnight at 4°C with the following primary antibodies: mouse anti-β-actin antibody, mouse anti-Bcl-2 antibody, mouse anti-Bax antibody, rabbit anti-BDNF antibody, rabbit anti-TrkB antibody, rabbit anti-CREB antibody, rabbit anti-p-CREB antibody (1:1,000; Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 hr with attempt secondary antibodies (1:2,000; Vector Laboratories), and ban detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).