Detection and characterization of NF-κB DNA binding activities in human neutrophils. (A) Neutrophils (5 × 106/mL) were cultured in the presence or absence of 1 μg/mL LPS, for 15 minutes at 37°C. Whole-cell extracts were prepared as described in Materials and Methods, and incubated in the presence or absence of DOC in the binding mixtures, before EMSA analysis using an oligonucleotide probe containing tandem repeats of the consensus NF-κB motif. The various DNA-binding complexes are indicated by the letters, A, B, and C. The amount of extract used in the binding reactions corresponded to 620,000 cell equivalents (approximately 20 μg of protein). (B) Neutrophils were cultured in the absence (first lane) or presence of LPS as indicated in (A), and whole-cell extracts (“WC”), as well as the corresponding cytoplasmic (“CYT”) and nuclear (“N”) extracts, were analyzed in EMSA. The amount of extract used corresponded to 550,000 cell equivalents (approximately 19 μg, 18 μg, and 2.2 μg for whole-cell, cytoplasmic, and nuclear extracts, respectively). (C) Whole-cell extracts from neutrophils cultured in the presence of 100 U/mL TNF-α (20 minutes at 37°C) were prepared and analyzed in EMSA. Before addition of the labeled NF-κB oligonucleotide probe to the binding mixture, extracts were incubated with a 10-, 25- or 50-fold molar excess of unlabeled NF-κB oligonucleotide (“cold”), or with an excess of unlabeled oligonucleotide containing mutated NF-κB binding sites (“mut”). The amount of extract used corresponded to 510,000 cell equivalents (approximately 16 μg of protein). (D) Neutrophils were cultured in the presence of LPS as described in (A), and nuclear extracts were analyzed in EMSA. Before addition of the labeled NF-κB oligonucleotide probe, extracts were incubated with antisera raised against p50 (no. 1141), p52 (no. 1267), c-Rel (no. 1136), the C-terminal region of p65/RelA (“C65”, no. 1226), the N-terminal region of p65 (“N65”, no. 1207), or without antisera (“-”). As a control, binding mixtures that only contained one of the antisera (in this case the anti-c-Rel, “Ab”), or normal rabbit serum (“NRS”), were routinely included on the gels. The amount of extract used corresponded to 960,000 cell equivalents, representing about 4.8 μg of protein. Supershifted complexes (“SS”) are indicated, as well as nonspecific bands (“NS”) present either in normal rabbit serum or in the control antiserum. Each of the experiments depicted in this figure is representative of at least four.
