Figure 6

K562 and K562/Adr cells show qualitative and quantitative differences in NFκB and AP1 DNA binding profiles. A) K562 and K562/Adr cells were pretreated with PMA (0.1 μg/ml) for 30 minutes. Nuclear lysates were analyzed for NFκB/DNA and AP1/DNA binding with a radiolabeled IL6 κB site- or AP1 motif-containing probe. Binding complexes formed were analyzed by EMSA. Loading of equal amounts of protein was verified by comparison with the binding activity of the repressor molecule recombination signal sequence-binding protein Jκ (RBP-Jκ). Specificity of the various complexes bound is demonstrated by supershift analysis with NFκB- and AP1-specific antibodies, as well as by competition with 100-fold excess cold oligonucleotide. B) K562 and K562/Adr cells were pretreated with 100 μM of quercetin, kaempferol, eriodictyol, WP283, or 6 μM of withaferin A for 2 h followed by incubation with PMA (0.1 μg/ml) for 30 minutes. Cell lysates were fractionated for cytoplasmic and nuclear extracts which were analyzed for NFκB, AP1, or Nrf2-dependent DNA binding with specific radiolabeled probes. Binding complexes formed were analyzed by EMSA.