Effect of IL-15 and IL-2 towards the activation of nuclear NF-κB DNA-binding activities in human polymorphonuclear neutrophils (PMN) and PBL. (A) Neutrophils were cultured for the indicated times (in minutes), and autologous PBL were cultured for 15 minutes in the presence or absence of 250 ng/mL IL-15 (ie, 20 nmol/L), 10³ U/mL IL-2 (∼5 nmol/L), or 100 ng/mL LPS. Nuclear extracts were prepared and analyzed in EMSA using a consensus NF-κB oligonucleotide probe. The amount of extract used in the binding reactions corresponded to 3.5 μg protein (about 3.1 × 10⁶ cell equivalents) for neutrophils, and to 1.5 μg protein (about 1.2 × 10 ⁶ cell equivalents) for PBL. This experiment is representative of six (neutrophils) and five (PBL). (B) Nuclear extracts from IL-15–activated neutrophils or autologous PBL were prepared as described in (A), and incubated without antibodies (–), or with specific antisera to p50, RelA, c-Rel, p52 and RelB, before the addition of labeled NF-κB probe and subsequent EMSA analysis. The amount of extract used corresponded to 3.5 μg of protein (neutrophils), and 1.0 μg of protein (PBL). This experiment is representative of three. Closed arrowheads indicate the inducible NF-κB complex; open arrowheads indicate supershifted bands.