Mouse beta actin antibody

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.

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).

Bax and Bcl-2 expressions were detected by western blot analysis, as described previously (Shin et al., 2016 (link)). The tissues were homogenized with lysis buffer with 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl·6H₂O, 1 mM EGTA, 1 mM PMSF, 1 mM Na₂VO₄, and 100 mM NaF, and then centrifuged at 14,000 rpm during 30 min. Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to measure protein content. Protein with 30 μg was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. As primary antibodies, mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology), and rabbit Bax antibody (1:1,000; Santa Cruz Biotechnology) were used. Horseradish peroxidase-conjugated anti-mouse antibody for beta-actin and Bcl-2 (1:3,000; Vector Laboratories, Burlingame, CA, USA), and horseradish peroxidase-conjugated anti-rabbit antibody for Bax (1:3,000; Vector Laboratories) were used as secondary antibodies. Experiment was performed in normal lab conditions and at room temperature except membrane transfer. Membrane was transferred at 4°C with the cold pack and prechilled buffer. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection.