Colorimetric protein assay kit

Following a previously described method [20 (link),21 (link)], Bax, Bcl-2, and cytochrome c expression was measured by western blot analysis. Using protein lysis buffer, hippocampal samples were lysed and a colorimetric protein assay kit was used to detect protein concentrations (Bio-Rad, Hercules, CA, USA). After 30 μg of protein was separated on sodium dodecyl sulfate-polyacrylamide gels, the separated protein was transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Anti-mouse β-actin antibody (1:1,000; Santa Cruz Biotechnology), anti-rabbit Bax antibody (Cell signaling Technology; 1:1,000), anti-mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology), and anti-rabbit cytochrome c antibody (1:1,000; Cell signaling Technology) were used as the primary antibodies. The secondary antibodies were horseradish peroxidase–conjugated anti-mouse antibodies (1:3,000; Vector Laboratories) for β-actin and Bcl-2, and horseradish peroxidase–conjugated anti-rabbit antibodies (1:5,000; Vector Laboratories) for Bax and cytochrome c. An enhanced chemiluminescence detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany) was used for band detection.

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).

Western blotting for the expressions of Wnt2, Wnt3a, Dkk1, and sFRP3 was performed, as previously described method (Cho et al., 2018 (link); Moon et al., 2018 (link)). Tissue samples harvested from the hippocampus were lysed in the protein lysis buffer containing 50 mM Tris-HCI (pH, 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% nonidet-P40 (NP40), 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 100-μm/mL leupeptin. Protein concentration was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein of 40 μg was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Anti-rabbit Wnt2 antibody (1:1,000; Abcam, Cambridge, MA, USA), anti-ribbit Wnt3a antibody (1:1,000; Millipore, Billerica, MA, USA), anti-ribbit Dkk1 antibody (1:1,000; Abcam), and anti-ribbit SFRP3 antibody (1:1,000; LSbio LSBio, Seattle, WA, USA) were used as the primary antibodies. For the secondary antibodies, peroxidase anti-rabbit IgG antibody (1:2,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used. Band detection was performed using as enhanced chemiluminescence detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany). Detected bands were calculated densitometrically using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD, USA).

Western blotting was performed, as the previously described method (Baek and Kim, 2016 (link); Kim et al., 2017 ). Tissue samples harvested from the hippocampus were lysed in the protein lysis buffer. Protein concentration was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein of 40 μg was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Anti-mouse β-actin antibody (1:2,000; Santa Cruz Biotechnology), anti-mouse Bax antibody (1:1,000; Santa Cruz Biotechnology), anti-mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibody. Horseradish peroxidase-conjugated anti-mouse antibodies (1:2,000; Santa Cruz Biotechnology) for β-actin, Bax, and Bcl-2 were used as the secondary antibody. Band detection was performed using as enhanced chemiluminescence detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany).

Western blot analysis for BDNF, TrkB, and AMPK was done as previously explained method [14 ,22 (link)]. Lysis buffer was applied to lyse hippocampal tissues, and protein content was detected by a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Sodium dodecyl sulfate-polyacrylamide gel was used to separated 30-μg protein, then reaction mixture was transferred to a nitrocellulose membrane, stopped reaction by applying dehydrated milk, and then incubated by primary antibodies. Mouse anti-β actin (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology), rabbit anti-TrkB (1:1,000; Santa Cruz Biotechnology), and rabbit anti-AMPK (1:1,000; Santa Cruz Biotechnology) were selected as the primary antibodies. Horseradish peroxidaseconjugated secondary antibodies were used, and enhanced chemiluminescence detection system (Santa Cruz Biotechnology) measured the expression of bands.

Western blotting for the determination of Bax, Bcl-2, BDNF, TrkB was conducted as previously described method (Park et al., 2019 (link); Song et al., 2018 (link)). The hippocampus tissues were homogenized on ice and lysed in a lysis buffer containing 50 mM Tris-HCl (pH, 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, and 100-mg/mL leupeptin. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein of 30 μg was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse β-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse Bax (1:1,000; Santa Cruz Biotechnology), Bcl-2 (1:1,000; Santa Cruz Biotechnology), and rabbit BDNF (1:1,000; Santa Cruz Bio-technology), TrkB (1:1,000; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse for β-actin, Bax, Bcl-2 and anti-rabbit for BDNF, TrkB were used as secondary antibodies.