Inhibition of endogenous TRAF2 by siRNA abolishes activation of NF-κB. (A) IBL-1, LCL-9001, and BCKN-1 cells were transfected with a NF-κB luciferase reporter plasmid and with either siRNA to TRAF1 (T1), TRAF2 (T2), TRAF3 (T3), TRAF5 (T5), or TRAF6 (T6), or scrambled (S) siRNA, or were mock transfected (−), and protein extracts were prepared 48 hours later. Luciferase assays were performed and values shown are averages (+ sem) of the combination of 2 independent experiments in which each transfection was performed in triplicate, and are presented as percentage change of relative luciferase values compared with untransfected cells. siRNA to TRAF2 resulted in marked reduction of NF-κB activity, but variable or no effect was seen on suppression of TRAF1, TRAF3, TRAF5, or TRAF6. The change of NF-κB activity in TRAF2 siRNA–transfected cells, compared with mock-transfected cells, was statistically significant (*P < .005). (B) To confirm effective and selective knockdown of each TRAF protein, immunoblot analysis using these extracts was performed using antibodies to the TRAFs indicated at the left of each panel. Actin reprobing was performed for all the blots to confirm even protein loading. (C) IBL-1 and LCL9001 cells were transfected with siRNA to the various TRAFs as in panel A at days 0, 2, and 4, and assessment of apoptosis performed by annexin V staining at day 6. Bars represent the average number of annexin V–positive cells (+ SEM) of an experiment in which each transfection and analysis was performed in triplicate.