NF-κB is required for IL-1β-induced MMP-9 expression. (A) Cells were pretreated with Bay11-7082 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with Bay11-7082, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with wild-type MMP-9 promoter and NF-κB-mutated MMP-9 promoter, and then incubated with IL-1β for 12 h. The promoter activity of MMP-9 was determined. (E) Cells were treated with IL-1β for the indicated times. The translocation of p65 was observed using a fluorescence microscope. (F) Cells were treated with IL-1β for the indicated times. The nuclear and cytosolic fractions were prepared and subjected to Western blotting using an anti-IκBα or anti-p65 antibody. Lamin A and GAPDH were used as a marker protein for nuclear and cytosolic fractions, respectively. (G) Cells were treated with IL-1β for the indicated times. The NF-κB promoter activity was measured. Data are expressed as mean±S.E.M. of three independent experiments. * P