SAA stimulates NF-κB activation.
(A) Neutrophils were stimulated with 2 μM SAA for different periods of time. Nuclear proteins were extracted and electrophoretic mobility shift assay was performed as described in “Materials and methods.” The NF-κB/DNA complex was detected by32P-labeled NF-κB oligonucleotide probe. Unlabeled NF-κB probe (100 × in excess) was used as a competitor to determine the specificity of DNA binding (lane 6). (B) Western blotting analysis was used to detect the cytoplasmic level of IκBα in neutrophils. Time-dependent degradation of IκBα was observed in SAA-treated (2 μM) but not the untreated neutrophils. Data shown are from one representative experiment of a total of 3, all with similar results. (C) HL-60 cells were transiently transfected with either a IL-8 luciferase reporter (−272–IL-8) or an NF-κB luciferase reporter (3 × NF-κB) as described in “Materials and methods.” The luciferase assays were performed after stimulation with 10 μM SAA for 4 hours at 37°C. Relative luciferase activities are expressed as fold induction over basal, after normalization of data against coexpressed β-galactosidase activities. (D) HL-60 cells (5 × 105 cells/200 μL) were cultured in serum-free medium with or without SAA for 4 hours. The secreted IL-8 was detected by ELISA. The fold increases of IL-8 production by the stimulated cells over unstimulated cells are shown. All data are presented as mean ± SEM, from 3 independent experiments, each performed in duplicate.
