Ecl reagent

Western blot analysis was performed, as previously described [24 (link)]. Initial cell lysis was used by RIPA buffer and protease/phosphatase inhibitors. Equal amounts of protein samples (25 or 50 µg) were separated to sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membranes by electrophoresis. The membranes were washed with 0.1% Tween 20 in TBS buffer (TBST) and were reactivated in blocking solution (5% bovine serum albumin (BSA)). The membranes were then incubated with anti-AMPK (Cell signaling; Danvers, MA, USA, 2532), phospho-AMPK (Cell signaling; 2535), brain-derived neurotrophic factor (BDNF, Abcam; Cambridge, UK, ab108319), tropomyosin receptor kinase B (TrkB, Cell signaling; 4603), phospho-TrkA (Tyr785)/TrkB (Tyr816) (Cell signaling; 4168), cAMP response element-binding protein (CREB, Cell signaling; 9197), phospho-CREB (Ser133) (Cell signaling; 9198), or β-actin (Santa Cruz Biotechnology; Dallas, TX, USA, sc-47778HRP) overnight at 4 °C. All primary antibodies except β-actin (1:5000 dilution) were used at a 1:1000 dilution. After repeated washing times with TBST, the membranes were reacted with HRP-conjugated secondary antibodies (1:5000 dilution). Protein detection was performed using an ECL reagent (Bio-Rad Laboratories, Hercules, CA, USA) and captured by Solo6S EDGE (Vilber Lourmat Sté, Collégien, France).

Protein lysates from LV tissue were prepared using RIPA buffer according to standard protocols. Primary antibodies against FGF receptor 1 (FGFR1) (rabbit polyclonal IgG, D8E4, Cell Signalling) and GAPDH (Millipore) were used. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Life Sciences), and bound antibody was detected using ECL reagent (Bio Rad). Quantification of Western blots was performed using ImageJ software. Protein expression was normalized to the expression of GAPDH.

M199 medium was purchased from Gibco Industries, Inc. (Auckland, NZ). Phosphate-buffered saline (PBS), sodium chloride (NaCl), bovine serum albumin (BSA), hematoxylin, and eosin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetramethylethylenediamine, protein assay kit, tween 20, ammonium persulfate, acrylamide, ECL reagent, and skim milk were purchased from Bio-Rad Lab. (Hercules, CA, USA). B-cell lymphoma-associated X protein (Bax), cytochrome c, and β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc., (Delaware Avenue, CA, USA). Cleaved caspase-3 and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The total glutathione (GSH) assay kit and the oxidized glutathione (GSSG)/GSH Quantification Kit were purchased from Dojindo Molecular Tech. (Tokyo, Japan). KOK was the same as that used in the previous study [14 (link)] in which chemical profiling and standardization of KOK had been performed and KOK (Lot No. SU12) was donated by Kwang Dong Pharmaceutical Co. (Pyongtaek, Korea).

The right parts of brain tissue were extracted in RIPA buffer plus protease/phosphatase inhibitors. Samples were immunoblotted as previously described [32 (link)]. Protein detection was carried out using an ECL reagent (Bio-Rad Laboratories) and visualized by ChemiDoc (Bio-Rad Laboratories). For quantification of relative protein expression, the optical density of the protein band of interest was normalized to the optical density of β-actin detected on the same membrane.

AR expression was analyzed using a previously described immunoblotting assay [44 (link), 45 (link)]. Briefly, protein extracts were subjected to SDS-PAGE and then transferred to a PVDF membrane (BioRad). The membrane was then incubated with the appropriate antibodies. After incubation, ECL reagent (BioRad) was applied to the membrane, and the signals were detected by Chemidoc XRS+ (BioRad) with a charge-coupled device (CCD) camera.