A role for Smad3/4 in the expression of Foxp3 in CD4⁺CD25⁺ T cells. (A) CD4⁺CD25⁺ T cells from healthy donors were transfected with the indicated siRNA and assayed for Foxp3 mRNA expression after 24 hours by FAST real-time RT-PCR (FAST TaqMan). GAPDH and Smad4 mRNA expression was also analyzed in parallel to monitor siRNA-mediated knockdown efficiency. Shown are results from 4 independent experiments using a different donor for each experiment. (B) To demonstrate Smad4 siRNA specificity, CD4⁺ T cells from 4 healthy donors were transfected with Smad4 siRNA or GAPDH siRNA (irrelevant control) and assayed for Smad2, Smad3, and Smad4 expression by FAST real-time RT-PCR (FAST TaqMan). (C) Schematic representation of the human Foxp3 promoter regions with putative transcription factor–binding sites.