IL-10 pretreatment of DCs inhibits IKK activity. (A) DCs were pretreated with 50 ng/mL IL-10 or left untreated for the indicated times, and then stimulated with 500 ng/mL LPS or 10 μg/mL anti-CD40 antibody for 15 minutes. DCs were pretreated with 50 ng/mL IL-10 or left untreated for 24 hours, and then stimulated with (B) 500 ng/mL LPS or (C) 10 μg/mL anti-CD40 antibody for the indicated times. (D) DCs were costimulated with IL-10 (50 ng/mL) and LPS (500 ng/mL) or treated with LPS alone for the indicated times. IKK signalosome was immunoprecipitated using anti-IKK1 or anti-IKK2 antibodies. In vitro kinase reactions were performed by preincubating immunoprecipitated protein with GST-IκBα and [γ-32P] ATP. Kinase reactions were analyzed using SDS-PAGE visualized by autoradiography. In all experiments, IKK1 and IKK2 expression was analyzed using Western blot. Data are representative of at least 3 independent experiments. The circled P indicates phospho.