2.9. Western Blot Analysis of NF-κB and MAPK8

The alterations of protein expression levels of NF-κB and MAPK8 were evaluated by Western blot analysis using the BioRad Mini Protean Tetra Cell wet transfer Western blotting system (Bio-Rad). Once treated with ACE (0.5–4 mM) or DMSO (1%) for 24 h, the cells were lysed using RIPA lysis buffer. Equivalent amounts of 30 μg protein were separated on polyacrylamide gel (10%) and transferred onto the polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 2 h at room temperature in skim milk (5%) in tris-buffered saline with Tween 20 (TBST) and incubated overnight with primary antibody diluted 1:1000 at 4 °C. Then, the blots were washed with TBST and incubated with a secondary antibody (anti-rabbit IgG, dilution of 1:5000) conjugated with horseradish peroxidase for 1 h at room temperature. JNK-bounded membranes were stripped using stripping buffer (Fisher Scientific Inc., Carlsbad, CA, USA) and then re-probed with β-actin primary antibody. After that, the bands were visualized using a luminol reagent by Kodak Molecular Imaging Software (Kodak Company, New York, NY, USA). ImageJ Software (National Institutes of Health, Baltimore, MD, USA) was used for densitometric analysis of the immunopositive band, and the results calculated as arbitrary units were normalized to the density of β-actin bands.