Effects of gene silencing of LANA by RNAi in BCBL-1 cells. (A) BCBL-1 cells were transfected with LANA siRNA or control siRNA or transfection reagent alone. Experiments were performed in duplicate. Forty-eight hours later, nuclear protein (20 μg) was subjected to Western blotting. Top panel: Western blot for LANA; bottom panel: Western blot for actin to demonstrate equivalent protein loading and the specificity of the LANA siRNA effects. (B) BCBL-1 cells were transfected with LANA or control siRNA and the indicated reporter plasmids. Protein was extracted after 48 hours for luciferase assays. The results are the means of 3 experiments and are reported as the relative reporter gene expression (in RLUs) of groups treated with LANA siRNA normalized to values for control siRNA ± SD. (C) The same protein used in panel A was used for Western blotting for c-Jun (top panel) and actin (bottom panel) to confirm equal protein loading. (D) EMSA with AP1 probe. The same nuclear protein was used as in panel A. Cold competition experiments demonstrated the specificity of the band. (E) BCBL-1 cells were transfected with LANA or control siRNA, and supernatants were harvested after 48 hours for IL-6 ELISA. The results are the means of 3 experiments ± SD.