Figure 2

Mutation of multiple Tax-1 or Tax-2B lysine residues leads to ubiquitination and sumoylation deficiency. 293T cells were cotransfected with vectors expressing wild type (WT) or lysine to arginine substitution mutants of Tax-1-6His and Tax-2B-6His, and with vectors expressing (A) HA-Ubiquitin or (B) HA-SUMO-1. Purification of the ubiquitinated and sumoylated forms was performed by Ni-NTA pulldown and the purified proteins were analyzed by Western Blot using an anti-HA antibody. (C) Comparable levels of wild type and mutant Tax proteins in the Ni-NTA pulldown preparations were ascertained by Western blot analysis using an anti-6His antibody. The abundance of the different species on the blots was estimated, and the percentages relatively to wild type species are indicated under each Western blot image.