Horseradish peroxidase conjugated anti mouse antibody

Western blot analysis was done, like the method described below [20 (link),21 (link)]. The Achilles tendon tissues were homogenized with lysis buffer containing 50mM Tris-HCl (pH, 8.0), 100mM NaF, 150mM NaCl, 1.5mM MgCl₂·6H₂O, 1mM EGTA, 1mM PMSF, 1mM Na₂VO₄, 10% glycerol, 1% Triton X-100, and then centrifuged for 30 minutes at 14,000 rpm. Rabbit CREB antibody (1:1,000; Abcam), rabbit phosphorylated CREB (p-CREB) antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit PKA antibody (1:1,000; Santa Cruz Biotechnology), rabbit phosphorylated PKA (p-PKA) antibody (1:1,000; Santa Cruz Biotechnology), mouse Bax antibody (1:1,000; Santa Cruz Biotechnology), mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology), and mouse β-actin antibody (1:1,000; Santa Cruz Biotechnology) were chosen as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories) for β-actin, Bax, Bcl-2, and anti-rabbit antibody (1:2000; Vector Laboratories) for CREB, pCREB, PKA, p-PKA were chosen as the secondary antibodies. Membrane transfer was carried out at 4°C, and all other steps were conducted at room temperature. The bands were calculated by Molecular Analyst version 1.4.1 (Bio-Rad Laboratories, Hercules, CA, USA).

Western blot analysis was performed, according to the previous method (Cho et al., 2018 (link); Wu et al., 2015 (link)). The right hemisphere was 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. Mouse GSK3β antibody, mouse phosphorylated GSK3β (p-GSK3β) antibody, mouse β-catenin antibody, and mouse phosphorylated β-catenin (p-β-catenin) antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories) for GSK3β, p-GSK3β, β-catenin, and p-β-catenin were used as secondary antibodies. Using a cold pack and prechilled buffer, membrane transfer was conducted at 4°C. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection.

Western blot analysis for NF-κB, IκB-α, Bax, Bcl-2, MMP-9 expression was used as explained below (Ko et al., 2020 (link); Park et al., 2020 (link)). Hippocampal tissues were lysed in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH, 7.5), 1 mM phenylmethylsulfonyl fluoride, 100-mg/mL leupeptin, 1% Nonidet P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate. Rabbit NF-κB antibody (1:1,000; Abcam), rabbit IκB-α antibody, rabbit phosphorylated IκB-α (p-IκB-α) antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit MMP antibody (1:2,000; Cell Signaling Technology, Inc., Danvers, USA), mouse Bax antibody, mouse Bcl-2 antibody, and β-actin antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories, Burlingame, CA, USA) for β-actin, Bax, Bcl-2, and horseradish peroxidase-conjugated anti-rabbit antibody (1: 2,000; Vector Laboratories) for NF-κB, IκB-α, and MMP were used as the secondary antibodies. Image-Pro Plus computer-aided image analysis system (Media Cybernetics Inc., Silver Spring, MD, USA) was used for band quantification.