Lack of induction of DNA-binding complexes by IL-10 in neutrophils. (A) PMN and autologous PBMC were incubated for 20 minutes in the presence or absence of 100 U/mL IL-10 or IFNγ. Cytoplasmic extracts were then prepared by nitrogen cavitation and analyzed in EMSA, using a 32P-labeled GRR oligonucleotide. For PMN extracts, 40 μg of protein was used in the binding reactions, whereas 10 μg of protein was used for PBMC extracts. This experiment is representative of at least 10. (B) PMN and autologous monocytes were incubated for 15 minutes with 100 U/mL IL-10 or IFNγ, and the resulting whole-cell extracts were analyzed in EMSA, using a32P-labeled hSIE/m67 oligonucleotide. Amounts of extract used are indicated. This experiment is representative of 2. (C) Characterization of the hSIE/m67-binding complexes induced in IL-10–treated monocytes. Purified monocytes were treated for 20 minutes with 100 U/mL IL-10, and whole-cell extracts were analyzed in EMSA. Binding reactions were performed in the presence or absence of specific anti-STAT antibodies as indicated, before the addition of the hSIE/m67 probe. This experiment is representative of 4.
