Western blotting.
For FOXP3 analysis on the protein level, 1 × 106 CD4+CD25– cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4–12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 °C. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3–31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),