In vitro GSI treatment blocks TGFβ1-induced Foxp3 expression and function. CD4⁺CD25⁻ splenocytes were isolated and stimulated under the following conditions: no treatment, + TGFβ1, + GSI alone, or + GSI + TGFβ1. (A) Notch1 expression in cells pretreated without or with GSI was evaluated by immunoblotting using antibodies that recognized the cleaved, active form of Notch1 (Notch1^IC). Antibody specific for HSP70 was used to control for loading. (B) Graphic representation of band intensities shown in panel A. (C) Effect of GSI treatment on Foxp3 expression was analyzed by flow cytometry using antibodies specific for CD4, CD25, and Foxp3. All plots are gated on live CD4⁺ cells. (D) Graphic representation of flow cytometry data from panel C. Data represent the means (± SD). **P = .001; *P < .05. (E) Foxp3 expression in CD4⁺CD25⁺ T cells treated without or with GSI was assessed by immunoblotting using antibodies specific for Foxp3. GAPDH was used as a loading control. (F) To assess the effects of GSI treatment on transcriptional regulation of foxp3, and its downstream target gene gpr83, total RNA was isolated from cells cultured under the conditions described in “RNA isolation and reverse-transcription–polymerase chain reaction” and analyzed by RT-PCR. Amplification of gapdh served as a loading control. (G) Cells were polarized as indicated in “Methods,” then washed, and cocultured at a 1:1 or 2:1 ratio with freshly isolated CD4⁺ T cells plus γ-irradiated APCs for 72 hours. *P < .05. Data are represented as mean suppression index (± SD). All results are representative of at least 2 independent experiments.