PI3K inhibitors block IKK activity and DNA binding of nuclear NF-κB. DCs were pretreated with either 200 nM wortmannin (Wo) or 50 μM LY294002 (LY) for 1 hour or left untreated, and then stimulated with 500 ng/mL LPS for the indicated times. (A) Cytoplasmic extracts were prepared, and phosphorylated Akt expression was analyzed using an antiphospho-Akt (Ser473)–specific antibody. The same membrane was reprobed with an anti-Akt antibody. IKK signalosome was immunoprecipitated using (B) anti-IKK1 or (C) anti-IKK2 antibodies, and in vitro kinase activity was measured as described. In all experiments, protein expression of IKK1 and IKK2 was analyzed through Western blot. (D) Nuclear extracts were prepared, and DNA binding to the H2K-specific oligonucleotide probe 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. The circled P indicates phospho.