Smad signaling, nodal expression, TGF-β production, and effects of follistatin on CD40L-mediated activin-A production by MoDCs. Immature MoDCs were cultured in 24-well plates for 4 hours with either GM-CSF + IL-4 alone or with CD40L (2 μg/mL), LPS (100 ng/mL), R-848 (1 μg/mL), or poly I:C (10 μg/mL). (A) DCs were then lysed, protein extracted, and Western blotting performed for detection of Smad 2/3, phospho-Smad 2, or β-actin. Data shown are one representative experiment from 3 separate donors. Nodal mRNA expression was assessed by quantitative RT-PCR in the same MoDC donors represented in Figure 1A-F. (B) Immature MoDCs were cultured in 96-well plates either with GM-CSF + IL-4 alone or with 2 μg/mL CD40L or 100 ng/mL LPS for 24 hours (C) or cultured in 96-well plates with 2 μg/mL CD40L together with increasing concentrations of follistatin (D). Supernatants were collected after 24 hours or at the times indicated and assayed for TGF-β (C) and/or activin-A (C,D) protein by ELISA. Data represent the mean plus or minus 1 SD of 3 separate donors. *P ≤ .01 versus CD40L only.