FIG 10.

Effects of Ikaros and R on each other's transcriptional activity. (A and B) Luciferase assays showing that R alleviates repression by Ikaros. 293T cells in 24-well plates were cotransfected with 70 ng reporter pGL4.15-c-Mycp (A) or pROM-Hes1p (B) and the indicated amounts of pcDNA3-HA-IK-1 (IK-1) and/or pcDNA3-R-V5 (R) plus pcDNA3.1 for total DNA of 200 ng per well. Luciferase activities were measured 44 h later, with assays performed in triplicate. Data were normalized externally to the basal activity observed for each reporter in the absence of R and IK-1. Immunoblots at the bottom of each panel show the relative levels of R and IK-1 present in these extracts. (C) Luciferase assays showing that IK-1 enhances, not inhibits, activation by R. EBV⁺ BJAB cells were infected for 2 days with lentivirus expressing IK-1 (IK-1) or the empty vector (Control). Subsequently, the cells were coelectroporated with 1.6 μg pCpGL-BALF2p and the indicated amounts of pcDNA3-R-V5 plus pcDNA3.1 for total DNA of 2.5 μg per 2.7 × 10⁶ cells. Luciferase activities were measured 48 h later, with assays performed in triplicate. Data were normalized internally to the amount of protein in each lysate and externally to the basal activity observed under each condition in the absence of R. Error bars show standard deviations. (D and E) Immunoblots showing that IK-1 synergizes with R and Z to induce high-level lytic gene expression. (D) 293T-EBV cells in 6-well plates were cotransfected with the indicated amounts of pcDNA3-HA-IK-1 and pcDNA3-R plus pcDNA3.1 for total DNA of 0.24 μg per well and harvested 48 h later. (E) BJAB-EBV cells were infected for 3 days with 525 lentivirus expressing IK-1 (IK-1) or 525 as empty vector (Control). Subsequently, the cells were coelectroporated with 0.8 μg pSG5-Z and/or pcDNA3-R-V5 plus pSG5 and pcDNA3.1 for total DNA of 2.5 μg per 2.7 × 10⁶ cells and were harvested 48 h later.