Assembly of Lys11-linked diubiquitin
(a) Domain structure of UBE2S, and autoubiquitination reactions with UBE2S wild-type and catalytic mutants. (b) UBE2S autoubiquitination occurs in cis. Wild-type UBE2S was mixed with GST-tagged inactive UBE2SC95A, and after precipitation of the GST-tagged protein, ubiquitination in supernatant (left) and precipitate (right) is analyzed. (c) Removal of the Lys-rich tail of UBE2S decreases autoubiquitination while preserving Lys11 specificity. (d) Purification of Lys11-linked diubiquitin by cation exchange chromatography. The integrated peak area (mAU*ml) is indicated. A gel showing protein-containing fractions is shown as an inset.