Impaired Foxp3 induction in cells expressing reduced levels of Notch1. CD4⁺CD25⁻ splenocytes from either wild-type (WT) or Notch1 antisense (AS) mice were stimulated with plate-bound αCD3ϵ plus αCD28 for 72 hours in the presence or absence of 2 ng/mL TGFβ1. (A) Cells were analyzed by flow cytometry using antibodies specific for CD4, CD25, and Foxp3. All plots are gated on live CD4⁺ cells. Plots are representative of 3 independent experiments. (B) Graphic representation of flow cytometry data from panel A. Data represented as the mean (± SD) of 3 samples per group. *P < .05. (C) Immunoblot detection of Foxp3, Notch1 intracellular domain (Notch1^IC), and GAPDH (loading control) from whole-cell lysates prepared from cells stimulated under the same conditions as in panel A. Data are representative of 2 independent experiments.