IL-10 pretreatment of DC inhibits IκB protein degradation, phosphorylation, and p65 phosphorylation. (A) DCs were pretreated with 50 ng/mL IL-10 or left untreated for 24 hours, and then were 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 anti-IκBα antibody or on stripping the membrane, antibody specific for IκBβ,IκBϵ,or β-actin. (bottom panels) Corresponding densitometric analyses were determined by measuring the ratio of intensity of the respective IκB proteins to β-actin expression per unit area for a given time and were presented as an arbitrary unit. (B) DCs were pretreated with 50 ng/mL IL-10 or left untreated for 24 hours, and then were stimulated with 500 ng/mL LPS or 10 μg/mL anti-CD40 antibody for 30 minutes. Cytoplasmic extracts were analyzed by Western blot using an antiphospho-IκBα antibody. The same membrane was reprobed with antiβ-actin antibody (bottom panel). Corresponding densitometric analyses determined as a ratio of intensity of phospho-IκBα to β-actin expression per unit area. (C) 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). Cytoplasmic extracts were analyzed for IκBα,IκBβ,IκBϵ,or β-actin expression. (D) 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. Nuclear extracts were examined through Western blot using antibodies specific for phospho-p65 and on stripping p65 protein or β actin. Densitometric analyses represent the ratio of intensity of phospho-p65 to β-actin expression per unit area and intensity of p65 compared with β-actin. The circled P indicates phospho.