Figure 4

Ubiquitination and sumoylation deficiency alters the intracellular localization of both Tax-1 and Tax-2B. 293T cells were cotransfected with vectors expressing wild type or mutant Tax-1 or Tax-2B and the transcriptional coactivator p300-HA. The cells were fixed, stained by triple immunofluorescence staining and analyzed by laser scanning confocal microscopy. Staining of Tax-1 expressing cells was with anti-Tax-1 IgG2a monoclonal antibody, anti-HA rabbit polyclonal antibody for the detection of p300-HA and anti-RelA IgG1 monoclonal antibody. For cells expressing Tax-2B, anti-Tax-2B rabbit polyclonal antibody and anti-HA monoclonal IgG2a antibody for the detection of p300-HA and the anti-RelA IgG1 monoclonal antibody were used. The secondary antibodies were goat anti-mouse IgG2a conjugated to Dylight 488, goat anti-rabbit IgG conjugated to Dylight 549 and goat anti-mouse IgG1 conjugated to Dylight 649. The profiles of the intensity of the fluorescence staining along lines crossing the nuclei are shown in the right panels (NE: nuclear envelope). The percentages of ubiquitination and sumoylation obtained in Figure 2 are indicated for each mutant (Ub/SUMO). White arrows point to cytoplasmic structures that contain Tax-1 and RelA at the boundary of the nucleus.