P ikkβ

RAW264.7 cells plated at a density 1.8 × 10⁶ cells/ml were incubated with LPS (100 ng/ml) at 37°C for 1 h. After that, the cells were washed extensively with PBS before being treated with CTP-NH₂ for 3 h at 37°C, followed by lysis of the cells. Cytoplasmic and nuclear protein fractions were obtained using NR-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc., New Zwaland). The protein concentrations were assessed with a CA kit (KeyGEN Biotech. Nanjing, China) according to the manufacturer’s instructions. Afterwards, total protein (40 μg protein/lane) was separated on 10% SDS-PAGE gels and then transferred to PVDF membranes (Bio-Rad). Next, the membranes were blocked with 5% non-fat dried-milk containing 0.05% TBST and then immunoblotted with specific primary antibodies against IKK-β, p-IKK-β, IкB-α, p-IкB-α, NF-кB (p65), p-NF-кB (p-p65), and β-actin (Santa Cruz, CA, USA). After being washed with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (HuaAn, Hangzhou, China). A ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) was used to quantify the density of the specific proteins.

Caspase-3 and caspase-8 antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Fas, DR3, DR4, DR5, DR6, IKKβ, p-IKKβ, IκBα, p-IκBα, p50, p65, Bcl-2, Bax, Histone-H1 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The cell culture materials were obtained from GIBGO^® of Introgen™ (Seoul, Korea).