Figure 1.

The catalytic cysteine of UBE2K is an in situ polyubiquitin chain building site. The figure shows UBE2K ubiquitination assays that were conducted for 30 min using Uba1, fluorescently labeled Ub⁸⁰⁰, and either wild-type UBE2K or UBE2K^K97R at 37°C as described in the Materials and Methods. Reactions were visualized by SDS-PAGE, which shows fluorescent ubiquitination products under reducing (left) and nonreducing (right) conditions. Products are identified as isolated polyubiquitin species (Ub_n) or polyubiquitin chains linked to the catalytic cysteine in UBE2K (UBE2K∼Ub_n) at the right of the gels.