Figure 3

The effect of EMS on IκB degradation and NF-κB transactivation in TNF-α-induced HDFs. HDFs were pre-treated with 250–500 μg/mL EMS for 3 h, and subsequently stimulated with 10 ng/mL TNF-α for 4 h. (A) IκB and p65 protein levels were analyzed by western blot. Nuclear p65 protein was analyzed using nuclear extracts. Lamin B was used as the loading control. The relative expression ratio of each band was calculated after densitometric analysis using image J software and normalization to β-actin or Lamin B signal. *p < 0.05 compared with control or TNF-α-treated group. (B) NF-κB transactivation activity was determined by luciferase assay using NF-κB reporter NIH-3 T3 cells. (C) The level of IL-1β mRNA was determined by real-time qPCR. (D) The level of IL-8 mRNA expression was determined by real-time qPCR. Independent experiments were performed in triplicate (*p < 0.05).