Angiotensin II enhances NF-κB DNA-binding activity and IκB degradation in human neutrophils. (A) Neutrophils (9 × 10⁶ cells/mL) were previously incubated in the absence (lanes 1, 2, 4) or presence (lane 3) of SB203580 (50 μM) for 30 minutes and then were treated with the indicated concentrations of angiotensin II (Ang) for another 30 minutes. Nuclear extracts were obtained, and EMSA was carried out as indicated in “Materials and methods.” Where indicated by an asterisk (lane 5), the binding reaction was performed in the presence of a 100-fold molar excess of unlabeled oligonucleotide under the same conditions as in lane 4. (B) Nuclear extracts from 100 nM Ang II–treated cells were preincubated for 30 minutes with, or without (lane 1), 1 μL antihuman p50 or p65 antibodies, as indicated, before the addition of the radiolabeled probe (lanes 2-4). (C) Cells were previously incubated in the absence or presence of PD098059 (50 μM) for 30 minutes (lanes 4, 6) or of CsA (1 μg/mL) for 60 minutes (lanes 3, 5) and then were incubated with Ang II (100 nM) for 30 minutes where indicated (lanes 2, 5, 6). Untreated controls are also shown (lane 1). Neutrophils were lysed, and nuclear extracts were used for EMSA analysis. (D) The cytosolic fractions (45 μg/lane) from cells incubated for 30 minutes with Ang II at the indicated concentrations were subjected to SDS-PAGE and then to immunoblotting analysis using an antihuman IκB-α antiserum. Data are representative of similar results of 3 independent experiments.