Figure 2.

Formation of polyubiquitin chains in the presence of GST-APC11. (A) Bacterial lysates expressing GST-APC10, GST-CDC26, and GST-APC11 were incubated with glutathione beads, were washed, were separated by SDS/PAGE, and were stained with Coomassie blue. (B) Identical amounts of GST-APC10, GST-CDC26, and GST-APC11 lysates as in A were bound to glutathione beads, were washed, and were used for in vitro ubiquitination. Samples were separated by SDS/PAGE (15 + 8%), were transferred to poly(vinylidene difluoride) membrane, and were immunodecorated with anti-ubiquitin antibodies. (C) Purified holo-APC, buffer QA, GST (750 ng), or the indicated amounts of GST-APC11 were incubated with E1, UBC4, and ATP as in B in the presence of ¹²⁵I-ubiquitin. Samples were incubated for 10 min at 37°C, were separated by SDS/PAGE, and were visualized by phosphorimaging.