2.6. NF-κB Reporter Assay

RAW 264.7 cells were inoculated in 12-well plates and transfected with a mixture containing the reporter gene pNF-κB-luc, the reporter constructs pRL SV40, and the Transfection Reagent for 24 h. After incubation with YD1 (2.5, 5, and 10 μg/mL) for 30 min, transfected cells were stimulated with LPS (1 µg/mL). The Luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) as described by Alam [21]. Immunocytochemistry and confocal image analysis of the NF-κB translocation were carried out as described by Ik-Soo Lee [24].