Notch3 increases D1 expression through modulation of Tal1 activity. (A-B) D1 expression analyzed by WB and RT-PCR from primary thymocytes derived from WT, Notch3-IC (N3-IC Tg), and N3-IC Tg/pTα^-/- mice (4 weeks of age). β-Tubulin and β-actin, respectively, were used as loading control. (C) M31 cells were transfected with either the pCDNA3 or Notch3-IC plasmids as indicated. Transient transfection efficiency, as assessed by GFP expression of a cotransfected expression vector, was greater than 70%. At 48 hours after transfection, total cell extracts were analyzed by immunoblotting with indicated antibodies. (C, bottom) Densitometric analyses of Tal1 level in M31 cells transfected with Notch3-IC. (D) M31 cells were transfected with the D1-promoter luciferase reporter plasmid plus expression vectors for constitutively active Notch3-IC, Tal1, and SP-1. Twelve hours after transfection, cells were treated with 25 and 50 μM PD98059, MEKK1 inhibitor, as indicated. Luciferase activity was determined at 24 hours after transfection. (E) SCB.29 cells were cotransfected with D1-Luciferase promoter, Tal1 and Sp1 expression vectors. After 12 hours from transfection, cells were treated with anti-CD3 for the time indicated. Luciferase activity was determined at 36 hours after transfection. (F, left) SCB29 cells were treated with anti-CD-3 Ab for 12 hours, and p-Tal1 expression was analyzed by Western blot, Tal1 blot was used as a loading control. (F, right) Densitometric analyses of pTal1 level in SCB29 cells treated with anti-CD-3.