Enhanced chemiluminescence detection kit

The PFC, striatum, hippocampus, and SN tissues were dissected and collected, and then were immediately frozen at −70°C. Western blot analysis was performed as previously described method (Ko et al., 2013 (link)). The sample 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 PMSF, 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 (Bio-Rad, Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. Rabbit GAPDH antibody (1:5,000; AbFrontier, Seoul, Korea) and mouse D₂ dopamine receptor antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody for D₂ dopamine receptor (1:2,000; Vector Laboratories) and anti-rabbit antibody for GAPDH (1:3,000; Vector Laboratories) were used as the secondary antibodies. The experiment was performed in normal lab conditions at room temperature except for membrane transfer. Membrane transfer was performed at 4°C with the cold pack and prechilled buffer. Band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).

Western blot was performed according to previously described methods [21 ]. Tissue samples harvested from the hippocampus were lysed in protein lysis buffer containing 50mM Tris-HCI (pH 7.5), 150mM NaCl, 0.5% deoxycholic acid, 1% nonidet-P40 (NP40), 0.1% sodium dodecyl sulfate (SDS), 1mM phenylmethylsulfonyl fluoride, and 100 µm/mL leupeptin. Protein concentration was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins of 40 µg were separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). The membranes were incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20, and then incubated overnight at 4℃ with the following primary antibodies: antimouse β-actin, antimouse Bcl-2, antimouse Bax, antimouse PI3K, antirabbit phospho-PI3K (p-PI3K), antimouse Akt, and antimouse phospho-Akt (p-Akt) (1:1,000; Santa Cruz Biotechnology). Subsequently, the membranes were incubated for 1 hour with secondary antibodies (1:2,000; Vector Laboratories), and band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). The bands were quantified using an Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA).

Western blot analysis for the NF-200 and BDNF was performed as the previously described method (Jung et al., 2016 (link); Kim et al., 2017 (link); Park et al., 2017 (link)). The sciatic nerve tissues were collected, and then were immediately frozen at −70°C. After homogenizing sciatic nerve tissues, tissue were lysed in a lysis buffer containing 50 mM HEPES (pH, 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium flouride. Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to determine protein content. Protein (30 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane.
For the primary antibody, mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology), mouse NF-200 antibody (1:1,000; Santa Cruz Biotechnology), and rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology) were used. As the secondary antibody, horseradish peroxidase-conjugated anti-mouse antibody (1:3,000; Amersham Pharmacia Biotechnology GmbH, Freiburg, Germany) for beat-actin and NF-200, and anti-rabbit antibody (1:2,000; Vector Laboratories) for BDNF were used. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for the band detection.