Figure 6.

Effect of melatonin and curcumin combination on IKKβ/NF-κB signaling. T24 cells were treated with melatonin (Mel, 1 mM) or curcumin (10 µM) alone or their combination. (A) After 48-h treatment, the binding of p65 and p50 to COX-2 promoter probe was detected via streptavidin-agarose pulldown assays in T24 cells. The protein levels of p65 and p50 in cytoplasm (B) and nucleus (C) were detected by western blot analysis. (D) At 48 h after treatment, we also observed the subcellular localization of p65 by confocal microscopy analysis. The percentage of NF-κB (p65) in cell nucleus was also quantified by counting the cell number of random five fields (control group was set as 100%). (E) At 48 h after treatment, the IKKβ, p-IKKβ, IκBα and p-IκBα proteins were analyzed by western blotting. (F) At 48 h after treatment, the IKKβ kinase activity was also detected in vitro using a cell IKKβ kinase activity spectrophotometry quantitative detection kit. The activity value and percentage was calculated by bringing into the formula. Results represent the mean ± SD of three experiments. ^*P<0.05, ^**P<0.01.