LMP2A suppression results in transcriptional down-regulation of TRAF2. (A) IBL-1 and LCL-9001 cell lines were transfected with siRNA to LMP1 (L1), LMP2A (L2A), or scrambled (S), or were mock transfected (−), and proteins were extracted 48 hours later. Immunoblot analysis was done with antibodies to TRAF2 and TRAF3. Actin reprobing was performed to assure even protein loading. (B) IBL-1 cells were transfected with siRNA to LMP2A (L2A) or scrambled (S), protein extracts were prepared after 48 hours, and immunoprecipitation (IP) using antibodies to LMP1 was performed. Pulled-down proteins were probed with antibodies to LMP1, TRAF2, and TRAF3 as indicated. (C) IBL-1 and LCL9001 cells were transfected with siRNA to LMP2A (L2A) or scrambled siRNA (S). RNA was extracted 48 hours after transfection, and quantitative real-time RT-PCR was performed in triplicate for LMP2A (L2A), TRAF2 (T2), TRAF3 (T3), and TRAF6 (T6) genes. Two independent experiments were performed for each cell line with similar results. The standard deviation (SD) for the triplicates was 0.1 or less and is not shown. The mean C_T value for both cell lines were calculated (avg C_T), and the normalized values (ΔC_T) were determined from corresponding GAPDH C_T values. The average ΔC_T values were calculated for scrambled and LMP2A siRNA–transfected cells. The difference between the 2 groups (ΔΔC_T) was used to determine the relative gene expression in LMP2A siRNA–transfected cells compared with scrambled siRNA–transfected cells. Efficiency of LMP2A suppression by siRNA is shown by the decrease of LMP2A RNA, which in turn resulted in a marked decrease of TRAF2 RNA, an increase in TRAF3 RNA in the LCL-9001 cell line only, and no change in TRAF6 RNA levels. The decrease of TRAF2 RNA was confirmed in the lymphoma cell line BCKN-1 after knockdown of LMP2A.