Anti-FOXP3 mAb, PCH101, was nonspecific for FOXP3 on activated CD4⁺ T cells. (A) Day 5-activated CD45RA⁺ cells treated with anti-TGFβ or with exogenous TGFβ1 were costained with PCH101 and 259D anti-FOXP3 mAbs or isotype controls. (B) Flow cytometric with 259D and real-time PCR analyses of FOXP3 protein and mRNA on unstimulated CD45RA⁺ cells (CD45RA + d0), unstimulated CD25^hi nTregs (CD25hi d0), day 5 non–TGFβ-treated CD45RA⁺ cells (CD45RA + d5 αTGFβ), day 5 TGFβ-treated CD45RA⁺ cells (CD45RA + d5 + TGFβ), and day 5-stimulated CD25^hi nTregs (CD25hi d5). Numbers in each quadrant as in Figure 1. (C) Flow cytometric analyses of FOXP3 with PCH101 and isotype control (shaded histogram) on CD45RA⁺ cells transfected with nonsilencing (NS, ——) or silencing (S, - - -) FOXP3 siRNA and activated for 5 days without TGFβ1 (left panel) or with TGFβ (center panel). Right panel is an overlay of TGFβ-treated CD45RA⁺ cells (- - -) and non-TGFβ-treated CD45RA⁺ cells (——) transfected with silencing FOXP3 siRNA. Data above are representative of 3 independent experiments.