Anti erk

Western blot analysis was performed as previously reported [15 (link)]. primary antibodies at 1:1000 for anti-ERK, -phosphorylated ERK (pERK), -Akt, -phosphorylated Akt (pAkt),—GSK3β, -phosphorylated GSK3β (pGSK3β), -GAPDH were purchased from Cell signaling Technology (Beverly, MA, USA), and anti-β-catenin was obtained from Abcam (Cambridge, MA, USA) overnight at 4°C. The ECL reagents (Amersham Corp., Arlington Heights, IL, USA) were used to detect the protein bands, and images were captured using the iBright^™ CL1000 imaging system (Thermo Fisher Scientific, Carlsbad, CA, USA).

The peripheral parts of muscle specimens were immediately frozen in liquid nitrogen and stored at −80°C for biochemical studies. Western blotting was performed as previously described (Fujimura and Usuki, 2015 (link), 2017 (link)). Briefly, the samples were sonicated for 5 s in tissue lysis buffer (T-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, USA) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, St Louis, USA). The samples were centrifuged (14,000 g for 1 h), and the supernatants were collected. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 μg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were then subjected to the following antibody probes: anti-MGF (Millipore, Billerica, USA); anti-IGF-I (Santa Cruz Biotechnology, CA, USA); anti-YAP1 (Novus Biologicals, Centennial, CO, USA); anti-phospho-YAP1 (Abcam, Cambridge, UK); anti-PAX7 (Cytoskeleton Incorporated, Denver, USA); anti-phospho-ERK, anti-ERK, anti-AKT, anti-phospho-AKT, anti-phospho-4EBP1, and anti-phospho-p70 S6 kinase (Cell Signaling Technologies); and anti-β-actin (Sigma-Aldrich).

Fermented Perilla frutescens (FPF), uracil, adenine, PCA, L7dGn, A7dGn and L7Gn were provided by Huons Co Ltd. (Seoul, Republic of Korea). Corticosterone, FXT and N-[2-[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl-benzo[b]thiophene-2-carboxamide (ANA-12) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Corticosterone enzyme-linked immunosorbent assay (ELISA) kit and dopamine ELISA kit were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Serotonin ELISA kit and ACTH ELISA kit were purchased from Abcam (Cambridge, UK). Anti-BDNF, anti-CREB, anti-pCREB, anti-ERK, anti-pERK, anti-TrkB, and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-pTrkB antibody was purchased from Abcam (Cambridge, MA, USA). All other chemical reagents were purchased from Sigma-Aldrich and were of analytical or HPLC grade.

Whole protein lysates were prepared using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Korea), and mitochondrial and cytoplasmic fractions were performed with a mitochondria isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by electrophoresis on 8–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, San Jose, NJ, USA). The membranes were blocked by incubation in blocking buffer (BD Biosciences) and probed with the following antibodies overnight at 4°C: anti-NeuN, anti-AT8, anti-Tau-5, anti-β-actin (Sigma–Adrich, St. Louis, MO, USA), anti-PSD95, anti-phospho(p)-Tau(T181), anti-p-Tau(S396; Abcam, MA, USA), anti-Drp1, anti-p-Drp1(S616), anti-COXIV, anti-GAPDH, anti-PARP, anti-cleaved caspase-3, p-Tau(S262), anti-CDK5, anti-ERK, anti-p-ERK, anti-GSK3β, anti-p-GSK3β(S9; Cell Signaling, MA, USA), and anti-p35 (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were washed with TBS with 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) for 1 h at room temperature. After washing with TBST, specific binding was detected using a chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA).

Standard Western-blot assays were used as described previously^{59 (link)}. The following primary antibodies were used: anti-PPARα (Abcam, Cata# ab24509), anti-ACLY (Santa Cruz, Cata# sc-517267), anti-FASN (Santa Cruz, Cata# sc-48357), anti-ACSL1 (cell signaling, Cata# 4047 S), anti-ACSL5 (Santa Cruz, Cata# sc-365478), anti-STAT3 (Santa Cruz, Cata# sc-8019), anti-pSTAT3 (cell signaling, Cata# 9145 S), anti-STAT1 (cell signaling, Cata# 14994 S), anti-pSTAT1 (cell signaling, Cata# 9177 S), anti-STAT4 (cell signaling, Cata# 2653 S), anti-pSTAT4 (cell signaling, Cata# 4134 S), anti-AKT (Santa Cruz, Cata# sc-5298, 1:2000 dilution), anti-pAKT (cell signaling, Cata# 9018 S), anti-pERK (cell signaling, Cata# 4376), anti-ERK (cell signaling, Cata# 9102), anti-pMAPK (cell signaling, Cata# 4511), anti-MAPK (cell signaling, Cata# 9212) and anti-β-actin (Sigma, Cata# A5441, 1:100,000 dilution) antibodies. Other than anti-AKT and anti-β-actin antibodies, the dilution for all other antibodies was 1:1000.

Retinas were homogenized, and 40 μg samples were resolved using a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which they were blotted to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk, incubated with primary antibodies and horseradish-conjugated secondary antibodies, and the signal was visualized with enhanced chemiluminescence. The following primary antibodies were used: anti-ERK (1:10,000; Cell Signaling Technology, Danvers, MA, United States), anti-pERK (1:2000; Millipore), anti- c-Jun N-terminal kinase (JNK, 1:2,000; Cell Signaling Technology), anti-pJNK (1:1,000; Cell Signaling Technology), anti-p38 (1:5,000; Abcam), anti-pp38 (1:10,000; Abcam), anti-Akt (1:10,000; Cell Signaling Technology), and anti-pAkt (1:1,000; Cell Signaling Technology). After the densitometric analysis, data were normalized against GAPDH (Millipore), and the ratio of protein expression in the treated eyes to the sham controls was calculated.