2.7. NF-κB Assay

BV2 cells were seeded at a density of 1.5 × 10⁶ cells/well in a 6-well plate and treated with NR, RR, and resveratrol, followed by LPS treatment for microglial activation and NF-kB translocation and transcription. The cells treated with the samples and LPS were incubated for 1 h and then harvested. Nuclear and cytosolic extracts from the harvested microglial cells were prepared using a Nuclear/Cytosolic Extraction Kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. The protein levels of NF-κB, histone-3, IkB, and p-IkB were evaluated by Western blot analysis. The expression of nucleolar and cytosolic NF-κB was measured using histone-3 and β-actin as loading controls, respectively. The expression of IkB and p-IkB was observed in the cytosolic fraction. The absence of β-actin expression in the nuclear fraction suggests the clear separation of nuclear and cytosolic fractions during fractionation without any contamination. Densitometry analysis of the bands was performed using ImageMaster™ 2D Elite software (version 3.1, Amersham Pharmacia Biotech, Buckinghamshire, UK).