FIG. 1.

Suppression of constitutive NF-κB activity inhibits overexpression of c-fos and AP-1 activity. (A) The expression of IκBα and IκBαM in MDAPanc-28/Puro and MDAPanc-28/IκBαM cells was determined by Western blot analysis with the cytoplasmic protein extracts using β-actin as loading controls. The nuclear protein extracts from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells were subjected to EMSA with NF-κB, AP-1, and the Oct-1 probe as a control. Expression of c-fos in MDAPanc-28/Puro and MDAPanc-28/IκBαM cells was determined by Northern blot analysis with GAPDH as the loading control. (B) Luciferase reporter gene assays for NF-κB and AP-1 activities were performed with wild-type (wt) and mutant (mt) NF-κB and AP-1 luciferase reporter gene constructs transiently transfected into MDAPanc-28/Puro and MDAPanc-28/IκBαM cells. (C and D) Competition and supershift assays were performed with EMSA to determine the specificity of the constitutive RelA/p50 NF-κB and AP-1 DNA binding activities. Ten micrograms of nuclear extract from MDAPanc-28/Puro cells was incubated with a 50× excess of unlabeled wild-type NF-κB or AP-1 probe (C and D, lane 2), mutant NF-κB or AP-1 probe (C and D, lane 3), and anti-NF-κB and anti-Fos antibodies (α-), with and without control peptides, as indicated. FP, free probe.