FIGURE 2.
K13 activates A20 promote through NF-κB pathway. A and B, the 293T cells were transfected with a control vector and a vector encoding FLAG-K13 (250 ng/ml) along with either A20-Luc or A20 mNF-κB-Luc or A20 m1-Luc reporter construct (75 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), and the reporter assay was performed as described under “Materials and Methods.” The values shown are averages (mean ± S.E.) of one representative experiment of three in which each transfection was performed in duplicate. Expression of the K13 was confirmed by Western blot analysis with an antibody against the FLAG epitope tag. C, K13-ERTAM induces A20 promoter activity upon treatment with 4OHT. The experiment was performed essentially as described in A. The values shown are averages (mean ± S.E.) of one representative experiment ofthree in which each transfection was performed in duplicate. D, wild-type K13 but not vFLIP E8- or NF-κB-defective mutants of K13 induces A20 promoter activity. 293T cells were transfected with the indicated FLAG-tagged expression constructs along with a A20 promoter luciferase construct. The experiment was performed as described for A. The values shown are averages (mean ± S.E.) of one representative experiment of three in which each transfection was performed in duplicate. Lower panel shows the expression of the transfected proteins as dermined by Western blotting with a FLAG antibody. E, dominant negative mutants of IκBα (IκBαΔN and IκBαS32A/S36A), IKK1 and IKK2 block K13-induced A20 promoter activity. The 293T cells were transfected either with an empty vector or K13, along with an A20 luciferase reporter construct and a reporter construct, as described in A. The amount of inhibitor plasmids (500 ng/well) was five times the amount of vector or K13 (100 ng/well) plasmid, and the total amount of transfected DNA was kept constant by adding empty vector. The values shown are averages (mean ± S.E.) of one representative experiment of three in which each transfection was performed in duplicate. Expression of the K13 was confirmed by Western blot analysis with the FLAG antibody. Asterisks indicate significance at levels of p ≤ 0.05. F, pharmacologic inhibitors of NF-κB block K13-induced A20 promoter activation. The 293T cells were transfected with an empty vector or a vector encoding K13 along with A20-Luc and pRSV/LacZ reporter constructs. Approximately 3 h after transfection, cells were treated with dimethyl sulfoxide (vehicle) or the indicated compounds for 18 h before cell lysis and measurement of reporter activities. Asterisks indicate significance at levels of p ≤ 0.05 compared with vehicle-treated cells.