Figure 4.

Ubiquitination reactions occurring in the presence of APC11 depend on E1, UBC4, ATP, and ubiquitin. (A) Coomassie-stained gel of reactions containing (+) or missing (−) the indicated components. All reactions were incubated for 30 min at 37°C, and missing components were substituted with buffer. (B) Autoradiogram of reactions separated under reducing (Left) or nonreducing (Right) conditions. All reactions were performed in the presence of E1, ATP, ¹²⁵I-ubiquitin, and with either UBC4, UBCx, CDC34, UBC2, GST-APC11, or combinations of these. Reactions were incubated for 5 min at room temperature, were stopped by addition of SDS/sample buffer with or without DTT, and were analyzed by SDS/PAGE.