Figure 2

Effects of Shc blockade on the secretion and mRNA expression of IL-6, IL-10 and IL-12 in DC maturation. Purified DCs were cultured with or without anti-mouse Shc mAb at various concentrations, as indicated, and stimulated with LPS (0.5 μg/ml) for 24 h and the IL-6 secretion was measured in triplicate supernatants by ELISA (A). DCs were transfected with Shc siRNA or negative control siRNA (NEG. siRNA) as described in the Materials and Methods. Quantitation of the RNAi efficiency for Shc silencing was analyzed by quantitative PCR to confirm the blockade effect of the Shc siRNA 24 h after transfection (B). The relative expression levels of IL-6 mRNA were measured in BMDCs stimulated by LPS in the absence or presence of Shc siRNA (50 nM), or NEG. siRNA (50 nM) for 24 h. Cells were collected, RNA was isolated, and IL-6 mRNA was determined by RT-PCR (C). Moreover, the amount of IL-10 and IL-12 were also determined by ELISA and RT-PCR. The data show enhanced secretion and mRNA level of IL-10 (D, E) and reduced production of IL-12 (F, G) from DCs in the presence of Shc siRNA. Each column represents the mean ± SE of the three independent experiments (n = 3; *, P < 0.05; **, P < 0.01).