*3.5. Western Blot*

Brain tissues were homogenized in lysis buffer (Cell Signaling Technology, Beverly, MA, USA) to collect the total proteins. Quantified proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane (Millipore, Temecula, CA, USA). The membranes were blocked with 0.1% Tween 20 in Tris-buffered saline containing 5% nonfat milk for 1 h at room temperature, and subsequently incubated with the primary antibody (Bcl-2, caspase-3, Bax, p-ERK, and ERK; Santa Cruz Biotechnology, Dallas, TX, USA; p-Akt, Akt, and β-actin; Cell Signaling Technology).

## *3.6. Polymerase Chain Reaction (PCR) Analysis*

The total RNA of brain tissues was extracted using the Hybrid-R RNA purification kit (GeneAll, Seoul, Korea), and converted to cDNA using the cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania). Amplification was performed in a DNA thermal cycler using the following synthesized primers: 5 -GATGAGGACCAGAAGGTTCG-3 (forward) and 5 -GATTGGGTA GTTCGGCATTG-3 (reverse) for BDNF; 5 -GCTGGGGCTCACCTGAAGGG-3 (forward) and 5 -GGATGACCTTGCCCACAGCC-3 (reverse) for GAPDH.
