Figure 3.

Characterization of ubiquitin conjugates formed in the presence of GST-APC11. (A) Purified GST (Left) or GST-APC11 (Right) were incubated for the indicated time with E1, UBC4, an ATP regenerating system, and ubiquitin as in Fig. 2B. Samples were separated by 12% SDS/PAGE. GST and GST-APC11 were detected by immunodecoration with anti-GST antibodies. The position of unmodified GST-APC11 is indicated by an arrowhead. (B) Identical samples as in A were separated by 15 + 8% SDS/PAGE and were stained with Coomassie blue. Identified proteins are indicated on the left (CPK, creatine phosphokinase; for all others, see text). The arrowhead indicates the position of GST-APC11. (C) A GST-APC11-mediated ubiquitination reaction as described in Fig. 2B was split into three aliquots as described in Materials and Methods. Identical amounts of total (T), beads (B), and supernatant (S) fractions were loaded on a 15 + 8% polyacrylamide gel and were transferred to poly(vinylidene difluoride) membrane. One part (Left) was decorated with anti-GST antibodies, the other part (Right) with anti-ubiquitin antibodies.