TZD18 inhibits NF-κB DNA-binding activities. Nuclear and cytoplasmic extracts were prepared from untreated and TZD18-treated (20 μm) SD1 cells after 24-, 48-, 72-, and 96-hour cultures. Equal amounts of protein were used for all assays. (A) EMSA analysis was performed to determine the DNA-binding capacity of NF-κB with the use of nuclear extracts. Competition analysis was performed by using a 100-fold excess of unlabeled NF-κB consensus-binding site. Antibodies specific for p50, p65, p52, cRel, and RelB were used in supershift analysis. Arrows indicate the positions of the super-shifted bands. The result is representative of 3 independent experiments. (B) Quantitative measurement of NF-κB activity was performed with nuclear proteins, as described in “Materials and methods.” NF-κB activity of TZD18-treated cells was expressed as a percentage of untreated control. Data represent mean ± SD of triplicate experiments. (C) NF-κB expression in nuclear extracts and phosphorylated I-κBα expression in cytoplasmic extracts were analyzed by Western blot. Membranes were blotted with antibodies specific for NF-κB subunit p65 and I-κBα phosphorylated at serine 32.