Fig. 4

H₂O₂ inhibits tumor necrosis factor alpha (TNFα)-induced activation of NF-κB. Rat brain capillary endothelial cells were activated with TNFα in the absence or presence of 2–5 µM H₂O₂ for indicated times (0–60 min). At the end of each incubation, cytoplasmic and nuclear extracts were prepared as described in Materials and Methods. Extracts were subjected to SDS-PAGE and immunobloted with antibodies directed either against IκB (cytocolic extracts, panels A and B) or against p65 subunit of NF-κB (nuclear extracts, panels C and D). (A) and (B) Representative results of Western blots. (B) and (D) Results of densitometry analysis of protein bands. Each data point represents mean ± SD of four to five experiments. “*” Marks significant difference between the treatments with and without H₂O₂ (ANOVA for repetitive measurements; p < .05). (E) and (F) Representative of electrophoretic mobility shift assays (EMSA) (n = 3). Nuclear extracts were prepared from untreated cells (control), or cells treated with TNFα alone (TNF) or together with 5 µM H₂O₂ (TNF + H₂O₂) and EMSA was performed as described in Materials and Methods. (F) The specific radioactivity associated with each band is presented.