K13-induced NF-κB activity is critical for the activation of CXCL10promoter. (a). Right panel: 293T cells were transfected with an empty vector, wild-type K13, K13-58AAA or MC159 (250 ng/well) along with a CXCL10 promoter-driven luciferase constructs (75 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), and the reporter assay performed as described under the Materials and Methods section. Left panel: Expression of the transfected proteins in the cell lysates is shown by immunoblotting with an antibody against the Flag epitope tag. Tubulin blot shows equal protein loading. (b). Right panel: Dominant-negative mutants of IκBα (IκBαΔN and IκBαSS32/36AA) block K13-induced CXCL10 promoter activity. 293T cells were transfected either with the indicated plasmids and reporter assay performed as described for Figure 3a. The amount of IκBα mutant 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. Left panel: Expression of the transfected dominant negative mutants of IκBα in the cell lysates is shown by immunoblotting with an antibody against IκBα. Tubulin blot shows equal protein loading. (c). 293T cells were transfected with an empty vector or a vector encoding K13 and subsequently treated with DMSO (vehicle) or the indicated compounds for 16 hours prior to cell lysis. Reporter assay was performed as described for Figure 3a.
Punj et al. BMC Medical Genomics 2009 2:50 doi:10.1186/1755-8794-2-50