FIG. 1.

cmvIL-10 activates Stat3 but not Stat1 in monocytes. A. Cell lysates from primary human CD14⁺ monocytes treated with 10 ng/ml recombinant hIL-10 or cmvIL-10 (R&D Systems) for 10 min were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with polyclonal antiserum (Cell Signaling) to detect phosphorylated or total unmodified Stat protein as indicated. Monocytes were isolated from buffy coats of healthy human donors by Ficoll density gradient centrifugation and magnetic separation with anti-CD14 microbeads (Miltenyi). B. Cells were pretreated with 10 μM JAK1 inhibitor {2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one, dissolved in dimethyl sulfoxide; Calbiochem} or MEK1 inhibitor (U0126; Cell Signaling) for 1 hour prior to treatment with 10 ng/ml cmvIL-10, and then lysates were immunoblotted. The total protein in each lysate was quantified (Bio-Rad protein assay kit), and 10 μg total protein was loaded in each well. Results are representative of experiments performed on cells from three different donors.