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Ligase activity. To test this, we expressed all of the Drosophila Roc proteins and chimeras as GST fusion proteins in E. coli and purified them (Fig. 6A). We then tested them for E3 ligase activity using a previously described in vitro assay that detects E2- and GST-Roc-dependent polyubiquitin formation in the absence of either Cullin or a particular substrate [24,26]. The ANBR protein was fully
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