IL-10 pretreatment of DC inhibits Akt phosphorylation. (A) 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). Cytoplasmic extracts were resolved through SDS-PAGE and analyzed by Western blot using an antiphospho-Akt (Ser473) antibody or, on stripping the membrane, antibody specific for Akt. (B) Under similar conditions, DCs were stimulated with 500 ng/mL LPS for the indicated times, and in vitro Akt kinase activity was measured by assessing phosphorylation of the GSK-3 fusion protein after SDS-PAGE resolution and visualization by autoradiography. (C) Ex vivo splenic DCs and 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. In vitro Akt kinase activity was measured. In all experiments, Akt protein expression was determined using Western blot. Data are representative of at least 3 independent experiments. The circled P indicates phospho.