Figure 6

Fusion of ubiquitin or SUMO-1 to the C-terminus of wild type or K1-10iR mutant modifies their intracellular localization and increases NF -κ B activity. (A) 293T cells were cotransfected with vectors expressing wild type or K1-10iR Tax-2B mutant, fused to ubiquitin (Tax-2-Ub and Tax-2 K1-10iR-Ub) or SUMO-1 (Tax-2-SUMO and Tax-2 K1-10iR-SUMO). The cells were fixed and stained by dual immunofluorescence staining with anti-Tax-2B rabbit polyclonal antibody and anti-RelA IgG1 monoclonal antibody. The secondary antibodies were goat anti-rabbit IgG conjugated to Dylight 549 and goat anti-mouse IgG1 conjugated to Dylight 649. The images were collected using a laser scanning confocal microscope. White arrows point to cytoplasmic structures at the boundary of the nucleus in which Tax-2-Ub and Tax-2 K1-10iR-Ub fusions colocalize with RelA. DIC, differential inference contrast. (B) 293T cells were cotransfected with 50 ng of vectors expressing wild type or K1-10iR mutant Tax-2B fused or not to ubiquitin or SUMO-1 and 250 ng of the NF-κB-Luc reporter construct. 50 ng of the phRG-TK Renilla Luciferase vector was added to normalize for transfection efficiency. Cells were lysed and the luciferase activity was measured in each extracts. The activity of each mutant fusion is expressed as percentages relatively to the equivalent wild type Tax-2-Ub or Tax-2-SUMO fusions. The values were normalized for equal quantity of Tax proteins in each extract. The values reported are the averages of three independent experiments.