Figure 5: Activation of NF-κB signaling. A. NF-κB-binding site/Luc reporter expression. Macrophage stably transfected with 5×NF-κB –binding site/ luciferase reporter were treated with SWCNTs for 16 h. Luciferase activity from cell lysates was measured. Data represent means ± SD from three samples. **, p<0.01. B. IκB degradation. Macrophage cells were treated with SWCNTs at 20 μg/ml for 30, 60, and 120 min. LPS (1 μg/ml) was used positive control. Cell lysate was immunoblotted with an anti-IκB antibody. Actin was used as a loading control. C. Fluorescent microscopy of nuclear NF-κB. Cells cultured in 8-well chamber slides were treated with LPS at 1 μg/ml for 5 h or with SWCNT at 20 μg/ml for 16 h. Cells were stained with an anti-NF-κBp65 antibody, followed by Alexia 488-conjugated second antibody. Image was taken under a confocal microscope. D. Binding of NF- κB to DNA. Cells were treated with LPS at 1 μg/ml for 5 h or SWCNTs at 20 μg/ml for 16 h. Binding of NF-κB to NF-κB-binding element was measured using nuclear extracts and the NF-κB p65 ELISA assay kit. NSB, non-specific binding **, p<0.01.