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Figure 2.
Fig. 2. E3-stimulated release of ubiquitin from the
UbcH5b-ubiquitin thioester. (A) WT and 31 mutants of UbcH5b were
tested for their ability to release ubiquitin from their
thioesters in the presence of buffer (-) or Apc2/11 (+) with
continuous E1-catalyzed ubiquitin charging. The bands
corresponding to free UbcH5b, UbcH5b-ubiquitin thioester, and
monoubiquitinated UbcH5b (with isopeptide-linked ubiquitin) are
indicated. (B) Same as in A except that CNOT4N was used as the
E3. (C) UbcH5b I88A was defective in supporting
APC/C^Cdh1-mediated ubiquitination of cyclin B1. Xenopus egg
APC/C was incubated with buffer or Cdh1 and assayed for its
ability to ubiquitinate an N-terminal fragment (residues 1-102)
of human cyclin B1 in the presence of the varying concentrations
of WT or mutant UbcH5b. Cyclin B-ubiquitin conjugates are
indicated.
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