IL-10 pretreatment of DC inhibits the DNA-binding activity of nuclear NF-κB. DCs were pretreated with 50 ng/mL IL-10 for the indicated times or were left untreated for 24 hours and then were stimulated with (A) 500 ng/mL LPS or (B) 10 μg/mL anti-CD40 antibody for 30 minutes. Nuclear extracts were prepared, and DNA binding to the H2K-specific oligonucleotide probe was analyzed using EMSA. (C) Components of the different NF-κB complexes binding to the H2K probe were determined through supershift analysis. (D), For the kinetics study, DCs were pretreated with 50 ng/mL IL-10 or left untreated for 24 hours and then stimulated with 500 ng/mL LPS for the indicated times (in hours). DNA binding was analyzed using EMSA. (E) DCs were costimulated with IL-10 (50 ng/mL) and LPS (500 ng/mL) or treated with LPS alone for the indicated times (in hours). (F) Isolated (ex vivo) splenic or cultured bone marrow– derived DCs were pretreated with 50 ng/mL IL-10 or left untreated for 24 hours and then stimulated with 500 ng/mL LPS for 30 minutes. Nuclear extracts were prepared, and DNA binding to H2K-specific oligonucleotide probes was measured using EMSA. A double-stranded OCT1 DNA probe was used as an internal control. Data are representative of at least 3 independent experiments. FP indicates free probe.