Expression of EBNA2 and LMP1 in EBV-transformed B-CLL cells after prolonged culture in vitro. B-CLL cells were cultured in the presence of irradiated J774A.1 cells for the period of time described in Figure 2. Four lymphoblastoid cell lines (LCLs EF3D, D-81, D-82, and LPK4) were generated by EBV infection of PBMCs from normal donors and were used as positive controls. Uninfected PBMCs from 2 B-CLL patients (CLL246 and CLL698) or 2 normal donors served as negative controls. For comparisons of mRNA levels of EBNA2 (A) and LMP1 (B), the cycle threshold (C_t) values of viral RNA (LMP1 or EBNA2) were subtracted by the C_t values of SETDB1 RNA. The 2^ − (viral RNA C_t − SETDB1 C_t) values were then normalized such that EF3D (an LCL) was 100% (because there were no detectable copies of EBNA2 or LMP1 in uninfected PBMCs). *Not detected. (C) For Western blots of LMP1, 5 μg each of the proteins extracted from cell lysates was loaded in each lane except for CLL493 (1.5 μg because of limited availability).