Figure 8

SUMO-1 fusion increases the cytosolic level and ubiquitination of Tax-P79AQ81A. (A) Confocal microscopy analysis showing the distribution of Tax or Tax-P79AQ81A fused or not to SUMO-1 (green) and of endogenous RelA (red) as well as nucleus staining (blue). The percentages of cells containing Tax in only nuclear bodies (NB), perinuclear spots (PS) or in both locations (NB + PS) are indicated. The percentages of cells containing RelA in the nucleus and the percentages of nuclear bodies-positive cells containing RelA in nuclear bodies are also indicated. At least 100 cells were analyzed in each condition. (B) Cell fractionation analysis. Cell extracts were separated in a cytosolic (C), intermediate (I) and nuclear (N) fraction as described [32]. Upper panel: Proteins were blotted with either an anti-Tax sera; anti-GM130, a marker of the intermediate fraction (Golgi apparatus), or anti-Lamins A/C, a marker of the nuclear fraction. Lower panel: the intensity of the Tax bands in all three fractions was quantified (Total Tax) and normalized to 100% for wt Tax. The proportion of Tax in the cytosolic fraction was calculated by dividing the intensity of each cytosolic Tax band by the value of Total Tax. (C) Ubiquitination of non-fused or fused wt Tax and Tax-P79AQ81A proteins. Ni-NTA experiments were performed in HeLa cells transfected with a control plasmid or the Tax-His constructs together with a HA-Ub-K63 plasmid. The percentages of HA-Ub-K63 conjugated products were normalized on the amount of unconjugated Tax and expressed in comparison to wt Tax (100%).