IL-10 pretreatment of DC inhibits phosphorylation of IKK1 and IKK2. DCs were pretreated with 50 ng/mL IL-10 or left untreated for 24 hours and then stimulated with (A) 500 ng/mL LPS or (B) 10 μg/mL anti-CD40 antibody for the indicated times. Cytoplasmic extracts were prepared, and the expression of phosphorylated IKK was analyzed using specific antiphospho-IKK1 (Ser180) or IKK2 (Ser181) antibody. The same membrane was reprobed with anti-IKK1 and anti-IKK2 antibodies. Data are representative of at least 3 independent experiments. The circled P indicates phospho.