Inhibition of endogenous LMP1 and LMP2A by siRNA results in depletion of constitutive NF-κB activity in EBV⁺ cells. (A) The indicated cell lines were transfected with a LMP1 siRNA (L1), and scrambled siRNA (s), and compared with mock-transfected cells (−). (B) IBL-1 cells were transfected with an LMP1 siRNA (L1), LMP2A siRNA (L2A), or both, and scrambled siRNA (s), and compared with mock-transfected cells (−). In panels A and B, protein extracts were prepared 48 hours after transfection and probed with antibodies to LMP1 or LMP2A, as indicated. Actin reprobing was performed to assure even protein loading. (C) BCKN-1, IBL-1, LCL-9001, RPMI-8402, and BC-3 cells were transfected with a NF-κB luciferase reporter plasmid, and either scrambled siRNA as a control (S), or siRNA for LMP1 (L1), LMP2A (L2A), or both. Luciferase assays were performed 48 hours after transfection. Values shown are averages (+ SEM) of 1 of 2 independent experiments in which each transfection was performed in triplicate, and are shown as relative luciferase units (RLU), which represent a ratio of NF-κB firefly luciferase to constitutive renilla luciferase. Baseline values correspond to those seen in mock-transfected cells and vary for each cell line.