Fig. 4.
No apparent TGFβ involvement in Foxp3 induction by PI3K and mTOR inhibitors. (a) Naive LN CD4 T cells were activated for 18 h in serum-free AIM-V medium and then exposed to TGFβ (1 ng/ml, 90 min, lane 1); TCR signal deprivation (90 min, lane 2); or TCR signal deprivation, rapamycin, and LY294002 (90 min for lane 3, 8 h for lane 4). Blots were sequentially probed with anti-pSmad2 (S465/467) and anti-Smad2/3. (b) Naive LN CD4 T cells activated as in a were deprived of TCR signals and TGFβ, and PI3K/mTOR inhibitors were added as indicated. Cultures were supplemented with neutralizing anti TGFβ (3 μg/ml) or the Smad kinase inhibitor SB431542. Foxp3 expression in the presence of anti TGFβ (dark gray bars) or SB431542 (light gray bars) was determined 2 days later and normalized to control cultures (black).