Notch1-IC, Notch2-IC, and EBNA2 exhibit profound differences in the regulation of the viral genes LMP1 and LMP2A and the classical Notch target genes HES1, HEY1, and DTX1. Cells were treated as outlined in Figure 1B and harvested at the indicated time points. (A) The regulation of LMP1 and LMP2A on mRNA level in the stably transfected cell lines was investigated by qRT-PCR. To compare LMP1 and LMP2A levels early after EBNA2 induction with that in proliferating EREB cells, RNA was also prepared 56 hours after estrogen addition and from cells that were continuously growing in the presence of estrogen (pr). mRNA levels of LMP1 and LMP2A were normalized to the expression of ribosomal protein genes and standardized to the value at the time point of induction (0 hours) to obtain fold inductions. Values are representative for 3 independent qRT-PCRs of 3 biologic replicates. Error bars represent SDs. (B) LMP1 and LMP2A protein levels at different time points after induction of Notch1-IC, Notch2-IC, or CAT, after EBNA2 reactivation or in proliferating CAT/EREB cells. Whole-cell lysates were harvested directly after estrogen withdrawal (0 hours), at the indicated time points after doxycycline or estrogen addition or from CAT/EREB cells continuously proliferating in the presence of estrogen (pr). Membranes were stained with α-LMP1 or α-LMP2A antibodies. Equal protein loading was controlled by tubulin staining. (C) The regulation of HES1, HEY1, and DTX1 in the stably transfected cell lines was investigated by qRT-PCR. Total RNA was harvested at the indicated time points after doxycycline or estrogen addition. mRNA levels of HES1, HEY1, and DTX1 were normalized to the expression of ribosomal protein genes and standardized to the value at the time of induction (0 hours) to obtain fold inductions. Values are representative of 3 independent qRT-PCRs of at least 2 biologic replicates.