(A) Post-sort TGF-β flow cytometry profile of TGF-β^m+ and TGF-β^m– cells. TGF-β^m+ cells were induced in mice using the tolerance protocol. On day 21, CD4⁺ T cells were prepared by negative selection, and cells expressing TGF-β on the cell surface (TGF-β^m+) were separated from those devoid of cell surface TGF-β (TGF-β^m–) using 2 rounds of sorting. The purity of the 2 populations was assessed by flow cytometry (~80% enrichment of TGF-β^m+ cells). (B) Experimental strategy. TGF-β^m+ or TGF-β^m– cells were mixed with DO11.10 TCR transgenic CD4⁺ T cells (target cells) in a 1:1 ratio and stimulated in vitro with whole OVA protein and APCs. (C) Staining with anti-Notch1 and KJ1-26 antibody or matching isotype control of an aliquot of 10⁵ total cells removed after 48 hours of incubation. Each dot plot contains approximately 1,000 events. Numbers in dot plots denote percentages. (D) Western blot analysis of the presence of cleaved Notch1 in total cell extracts (TCEs) prepared from the mixed cultures after 24 hours of incubation using antibody specific to cleaved Notch1. Expression of β-actin is shown as a marker for protein loading. (E) Analysis of HES1 expression by immunoblotting of nuclear extracts (NEs) prepared after 72 hours of culture. CREB-1 expression was examined to assess protein loading. Results shown are representative of 2 independent experiments.