In vitro GSI treatment blocks the binding of Notch1, CSL, and Smad to the foxp3 promoter without inhibiting histone acetylation. Chromatin immunoprecipitation (ChIP) of CD4⁺CD25⁻ splenocytes stimulated with plate-bound αCD3ϵ plus αCD28 and 2 ng/mL TGFβ1 in the presence or absence of GSI for 24 hours. (A) Primer sets were designed to span putative CSL and Smad binding sites within the foxp3 promoter. Rabbit αNotch1, rabbit αCSL, and mouse αSmad1/2/3 were used to immunoprecipitate protein-DNA complexes. De–cross-linked DNA was amplified by PCR using either primer set 1 (B) or primer set 2 (D). Band intensities were calculated using ImageJ software, version 1.38 (National Institutes of Health, Bethesda, MD; panels C,E). Data are representative of 3 independent experiments. (F) ChIP assay using antibody specific for acetylated histone H3 (αacetyl H3) and either primer set 1 from panel A or the region of the Foxp3 enhancer containing previously identified Smad3 and NFAT binding sites. Data are representative of 2 independent experiments. Input indicates total chromatin. No Ab (beads only), rabbit IgG, and mouse IgG were used as isotype controls.