pTα is required for optimal expansion of E2A^–/– lymphomas. (A) FACS analysis of E2A^–/– lymphoma (1.F9) with control (black histogram) or pTα siRNA (gray histogram) virus and stained with anti–TCR-β antibody (solid line) or control IgG (broken line). Anti–TCR-β staining is shown on GFP⁺ cells. (B) Northern blot of RNA isolated from GFP⁺ lymphomas 48 hours after infection with control or pTα siRNA virus. The blot was probed with pTα or actin cDNA, as indicated. (C) The 70Z/3 pre–B cell line (♦), E2A^–/– lymphomas 0531 (•) and 1.F9 (▴), and the Notch3-transformed cell line N3T (▪) were infected with control (open symbols) or pTα siRNA (filled symbols) virus, and the percentage of GFP⁺ cells was determined every 24 hours by flow cytometry. Infection efficiency for each cell line was 40% to 60%. (D) Control (▦) and pTα siRNA virus–infected (▪) cells were incubated with DHE for 30 minutes starting 48 hours after infection. The percentage of GFP⁺ cells showing increased fluorescence with DHE is shown. (E) Western blot of whole cell extracts made from lymphoma lines treated for 48 hours with DMSO (–) or GSI (+) or 48 hours after infection with control virus (B) or pTα siRNA–producing virus (S). The blots were probed sequentially with antibodies detecting p21 or p27. Total protein loading was visualized before hybridization using Amido Black and mirrored the intensity of the nonspecific band present on the p21 blot.