A) Naïve primary T cells were stimulated with anti-CD3/CD28, with or without TGF-β, IL-6 and CIP for 72hrs. Whole cell protein lysates were prepared from these samples and western blot analyses were performed to probe for p-Smad2 (Ser465/467), p-Smad3 (Ser423/425) and total Smad2 and total Smad3. B) Similarly, both Cdk5+/+ T cells and Cdk5−/− T cells were stimulated and treated under conditions noted in (A) and probed for the presence of p-Smad2/3 and total Smad2/3. C) Luciferase activity was examined in EL4 T cells transfected with the Smad-binding element (SBE) reporter construct and E) the Foxp3 promoter and enhancer I reporter construct. Cells were stimulated with anti-CD3/CD28 with or without TGF-β, IL-6 and CIP as indicated.