Activation of STAT3 phosphorylation by murine and viral IL-10 produced in tobacco plants. STAT3 phosphorylation (STAT3-YP) was assessed by immunoblot analysis on protein extracts of macrophage cells treated with increasing doses of plant-derived IL-10. (a) J774 cells were cultured for 15 min in the presence of eluate from wild type tobacco plants (lane 1; protein amount equivalent to the 150 ng/ml sample of the mIL-10-producing tobacco line), 20 ng/ml of commercial mIL-10 produced in insect cells (lane 2) or increasing concentrations of plant-derived mIL-10 (lanes 3–6); (b) J774 cells were cultured for 15 min in the presence of the eluate from purification of wild type tobacco plants (lane 1; protein amount equivalent to the 500 ng/ml sample of the viral IL-10-producing tobacco line), 150 ng/ml of commercial vIL-10 (lane 2) or increasing concentration of plant vIL-10 (lanes 3–6). Total cell extracts (50 μg) were separated by SDS-PAGE and immunoblots were performed as described in the Experimental Procedures section. One experiment representative of two is shown. The blots were scanned on the Odyssey Infrared Imaging System at 700 and 800 nm. The relative STAT3-YP levels, as quantified by the Odyssey software and normalized for the total STAT3, are reported below each panel.