Tal1-sustained expression depends on IL-2/IL-2R pathway. (A) Fluorescence-activated cell sorting (FACS) analysis of CD25 and CD122 expression in thymocytes derived from WT Notch3-IC Tg and N3-IC Tg/pTα^-/- mice. (B, left) Tal1-luciferase promoter activity in Notch3-overexpressing N3-232T cells. (B, right) GAL4-luciferase control promoter activity in N3-232T cells. Cells were transfected with either Tal1-promoter luciferase or GAL4-luciferase responsive promoter construct, 0.25 μg/well in 24-well dishes, and treated with the indicated amount of IL-2 for 12 hours; cells were harvested 24 hours after transfection for luciferase assay. All conditions were tested in triplicate samples, and SD is indicated. (C-D) Analysis of Tal1 protein expression by Western blot, in N3-232T, SCIET27, and SCB29 cells treated with the indicated amount of IL-2 for 24 hours; β-tubulin was used as loading control. (E) 232 cells were cultured with (+) and without (-) neutralizing antibodies anti-CD25 (5 μg/mL) alone or with added IL-2 (100 U). mAb was added 2 hours before addition of the cytokine, and the cells were collected after 24 hours. (F) Sample in lanes 1 and 2 of panel E were probed with p-Tal1 and β-tubulin antibodies.