Effect of LPS, TNF-α, and fMLP towards NF-κB activation in human neutrophils. (A) Cells (5 × 106/mL) were cultured at 37°C in the presence or absence of 1 μg/mL LPS, 100 U/mL TNF-α, or 10 nmol/L fMLP for the indicated times (in minutes), and nuclear extracts were prepared and analyzed in EMSA. The amount of nuclear extract used in the binding reactions corresponded to 106 cell equivalents (upper panel) and 1.5 × 106 cell equivalents (lower panel), representing approximately 4.5 and 7.0 μg of protein, respectively. (B) Neutrophils were cultured in the presence or absence of 100 U/mL TNF-α, 10 ng/mL IL-1β, 10 nmol/L fMLP, 10 ng/mL PMA, or 10 nmol/L LTB4 as described in (A) for the indicated times (in minutes). Nuclear extracts were then prepared and analyzed in EMSA. The amount of nuclear extract used corresponded to approximately 750,000 cell equivalents. Each of the experiments depicted in this figure is representative of at least four.
