Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

eji2262-sup-0001-s1.pdf96KSupplemental Figure 1. Identification of two nuclear export sequences in FOXP3. (A) PCR primer sequences used for cloning of the different GFP-FOXP3 mutants and isoforms GFPFOXP3 2, GFP-FOXP37 and GFP-FOXP327. (B) Exchange of amino acids within the localization signals by site directed mutagenesis. Numbering of amino acids corresponds to the full-length human FOXP3 protein. m, mutated.
eji2262-sup-0001-s1.pdf96KSupplemental Figure 2. (A) Purity of flow-sorted human CD4+CD25- and CD4+CD25+ T cells. Cells were surface stained with anti-CD4-APC and anti-CD25-PC5. (B) Purity of flowsorted CD4+CD25+CD45RA+ and CD4+CD25+CD45RA- T cells.
eji2262-sup-0001-s1.pdf96KSupplemental Figure 3. Changes in FOXP3 protein expression level (MFI) (A) and percentages of FOXP3 expression (B) in CD4+CD25- and CD4+CD25+ T cells stimulated with CD3/CD28 beads for 8 days and analyzed by flow cytometry at indicated time points. (mean ± SD, n = 6). (C) At day 2 of the primary culture activated CD4+CD25- T cells and freshly isolated CD4+CD25+ regulatory T cells were added to a secondary mixed lymphocyte reaction, consisting of CFSE-labeled CD3+ effector T cells and allogeneic stimulator APC. CFSE dilution profile showing the relative suppression of Teff proliferation at a Treg:Teff ratio of 1:1 (data representative of three equivalent experiments; gated on live CD4+ and CFSE+ cells).
eji2262-sup-0001-s1.pdf96KSupplemental Figure 4. Sequence alignment for nuclear export sequences NES1 and NES2 of FOXP3 from different species with ClustalW2.

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