Chemiluminescent detection reagent

Whole lysates were harvested using a Radioimmunoprecipitation Assay (RIPA) buffer containing protease inhibitor cocktail (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail set III (Calbiochem, CA, USA). Nuclear extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA.). Equal amounts (20 μg) of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ, USA), which were blocked with 5% skim milk in TBS/T (Tris buffered saline in 0.1% TWEEN® 20) buffer for 1 h. The membranes were then treated overnight with antibodies specific to iNOS, NF-κB p65, HO-1, NRF2, phospho-CREB, CREB, phospho-p38 (Thr180/Tyr182), p38, phospho-Erk (Thr202/Tyr204), Erk, phospho-JNK (Thr183/Tyr185), JNK, phospho-Akt (Ser473), and Akt (Cell Signaling Technology, Beverly, MA). Blots were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h at room temperature. HRP was detected using a chemiluminescent detection reagent (Amersham Biosciences). β-actin (Sigma-Aldrich) and proliferating cell nuclear antigen (PCNA; Cell Signaling Technology) were used as loading controls. Chemiluminescence was visualized using an LAS-3000 LuminoImage analyzer (Fujifilm, Tokyo, Japan) [23 (link)].

The hippocampus was homogenized in 300 μl lysis buffer (Pro-Prep™; Intron Biotechnology, Korea) containing 1 mM PMSF and 1 μg·ml⁻¹ of protease inhibitor mix. Equal amounts (20 μg) of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were then blocked in 5% skim milk in Tris-buffered saline with 0.1% TWEEN® 20 (TBS/T) for 1 h. The membranes were probed overnight with antibodies for brain-derived neurotrophic factor (BDNF; Abcam, Cambridge, UK), phosphorylated cAMP response element-binding protein (p-CREB), and CREB (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C. Next, blots were incubated in horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h at 25°C, and HRP was detected using a chemiluminescent detection reagent (Amersham Biosciences). β-Actin (Sigma-Aldrich) was used as a loading control [24 (link)].