Effect of plant-derived murine IL-10 on LPS-induced SOCS3 mRNA and SOCS3 protein expression. (a) J774 cells were stimulated for 2 h with 100 ng/ml of LPS alone or in combination with commercial mIL-10 (20 ng/ml), or plant mIL-10 (50 and 100 ng/ml). Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Total RNA was extracted and then analyzed for SOCS3 mRNA expression by real time RT-PCR. The graph shows the SOCS3 mRNA levels (mean ± SD) assayed in triplicate and normalized to GAPDH expression. (b) J774 cells were incubated for 18 h in the presence of LPS alone (lane 2) or in combination with 20 ng/ml commercial IL-10 (lane 3) or 50 ng/ml (lane 4) or 100 ng/ml (lane 5) plant derived mIL-10. Non stimulated cells ('medium') were included in the analysis to determine SOCS3 basal expression levels. Whole cell extracts (50 μg) were immunoblotted using anti-NH2 terminus SOCS3 antibody (upper panel) and antibodies specific for GAPDH (lower panel), followed by incubation with AlexaFluor 680 goat anti-rabbit and IRDye 800 goat anti-mouse antibody. The relative levels of SOCS3 protein, as quantified by the Odyssey software and normalized for the total GAPDH content, are reported at the bottom of each lane. The data shown in (a) and (b) are representative of two independent experiments. ** p < 0.001.