Figure 1

NFκB activity and cytoplasmic and nuclear levels of IκB and NFκB proteins during persistent neutrophil stimulation with LPS and TNFα. (A) Neutrophils were stimulated with LPS (100 ng/mL) or TNFα (10 ng/mL) for 0, 0.5, 3, 6, 9, and 12 h, and NFκB activity was measured in nuclear extracts by EMSA. (B) Supershift analysis of NFκB complexes induced in human neutrophils after 9-h stimulation with LPS and TNFα. Nuclear extracts prepared from neutrophils stimulated with LPS (100 ng/mL) and TNFα (10 ng/mL) for 9 h were incubated with NFκB antibodies specific against p50, p65, and c-Rel subunits. (C) Neutrophils were stimulated with LPS (100 ng/mL) or TNFα (10 ng/mL) for 0, 0.5, 3, 6, 9, and 12 h, and the cytoplasmic (CE) and nuclear (NE) levels of IκBα, IκBε, and p50 and p65 NFκB proteins were analyzed by immunoblotting. The presence of cytoplasmic proteins in the nuclear fraction was monitored using lactate dehydrogenase (LDH) antibody. Nuclear contamination in the cytoplasmic fraction was assessed using lamin B-specific antibody. Each lane contains approximately 5 × 10⁵ cells. (D) Densitometric evaluation of IκBε levels in stimulated human neutrophils. The IκBε bands were scanned and the densities were normalized to densities of p21 used a loading control. The values at T = 0 for CE and NE were arbitrarily set to 1, and the other values are presented relative to those values. The data represent the means of five independent experiments ± SE.