TCR and CD28 stimulation of human CD4+CD25- T cells produced TGF-β and exhibited phosphorylation of Smad2. A. CD4+CD25- T cells (1 × 106/ml) were cultured with anti-CD3 and anti-CD28 in X-Vivo 20 serum-free medium for the indicated time points. Cell-free supernatants were either untreated (for active TGF-β) or treated with 1 N HCl (for total TGF-β) followed by ELISA for TGF-β1 measurement. The values are shown as Mean ± SD of individuals in each group at each time point (n = 3 to 9). B. The relative ratio of active to total TGF-β is shown in each time point as in A. C. Western blot analysis of P-Smad2 in cultured CD4+CD25- T cells (72 hrs). Whole cell lysis protein (70 μg/ml) was loaded into each lane. P-Smad2 was detected with anti-P-Smad2 antibody. α-tubulin was used as host protein control. 3+28: anti-CD3+anti-CD28; Med: medium.