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Fig. 3. In vitro C. elegans ubiquitination assays with APC-11 and UBC-2. Ubiquitination reactions were carried out as described in Materials and Methods. (A) Reactions were performed with GST-APC-11 and UBC-2. Ubiquitination reactions were probed with anti-HA antibody. Activity depends on the presence of both E1 and E2 enzymes, as well as APC-11. In the last lane, C. elegans RBX-1 is used as a positive control with the human E2, UbcH5c. (B) APC-11 that contains a mutation in a conserved residue of the RING finger shows no ubiquitination activity. The Coomassie blue-stained gel on the right shows purified wild-type and mutant GST-APC-11 fusion proteins.





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