Figure 3

The effect of H₂O₂ on the TNFα-induced phosphorylation and degradation of IκBα, and the nuclear translocation of p65. (A) HEK293 cells were treated with various doses of H₂O₂ for 20 min and stimulated with TNFα (20 ng/ml) for 10 min. The levels of IκBα and phosphorylated IκBα (p-IκBα) in the cytoplasmic extract (CE) and p65 in the nuclear extract (NE) were determined by immunoblotting analysis using specific antibodies. β-Actin and nucleoporin (Nucln) were used as control proteins for the cytoplasmic and nuclear extracts, respectively. (B) The cells were exposed to H₂O₂ (0.5 mM) for 20 min and stimulated with TNFα for various times. The changes in the levels of proteins in the cytoplasmic and nuclear extracts were determined by immunoblotting analysis as described in (A). (C) Nuclear extracts prepared from cells treated as in (B) were used to determine NF-κB-DNA binding activity by EMSA. The upper panel shows the retarded NF-κB band, and the lower panel shows unbound free probe.