Activity of AP1 and Stat5 in naive CD4⁺ and CD4⁺CD80^{acq hi} T cells and effect of endogenous IL-2 on NFκB, AP1, and Stat5. AP1 and Stat5 activity in nuclear extracts from CD4⁺CD80^{acq hi} T cells was investigated using EMSA. (A) The CD4⁺CD80^{acq hi} T cells were separated from PCC-pulsed P13.9 APCs (0 hour) and further cultured at various time points in the absence of APCs and PCC peptide, and AP1 activity in these cells was observed. (B) Binding band of AP1 in the cells mentioned in the discussion of Figure 5A was analyzed using competitive and supershift assays. In the competitive assay (left), 50-fold of cold AP1 and NFκB oligmers were used to compete with labeled AP1 probe. In the supershift assay (right), different anti-AP1 Abs were used to determine the major subunit of AP1. (C) CD4⁺CD80^{acq hi} T cells were cultured in the absence or presence of exogenous PCC (1 μg/mL) without APCs for 6 or 24 hours. Binding activity of AP1 was measured. (D) CD4⁺CD80^{acq hi} T cells were separated from PCC-pulsed P13.9 fibroblasts (0 hour) and further cultured in the absence of APCs and PCC peptide or in the presence of 1 μg/mL PCC peptide at various times, and Stat5 activity in these cells was determined. (E) Stat5 binding band was identified using supershift assay with anti-Stat5 Ab. (F) Naive CD4⁺ T cells were cocultured with PCC-pulsed P13.9 fibroblasts for 20 hours. After the separation from P13.9 APCs, CD4⁺CD80^acq T cells were cultured in fresh medium in the absence or presence of anti-IL-2 neutralizing Abs (10 μg/mL) at various time points. Activity of NFκB, AP1, and Stat5 in CD4⁺CD80^acq T cells was measured using EMSA.