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Figure 4. Xnf7 is a ubiquitin ligase and its ligase activity is required for its effects on mitotic exit. (A) Xnf7 contains a RING finger consensus sequence. The eight amino acids in bold are the zinc-binding sites of the RING finger. Xnf7WT, Xnf7 mutant 3 (Xnf7m3) (C160A), and Xnf7 mutant 36 (Xnf7m36) (C160A and C168A) were produced in bacteria. (B) Xnf7 has ubiquitin ligase activity. Xnf7WT (lane 1), Xnf7m3 (lane 2), or Xnf7m36 (lane 3) were incubated with purified E1, UbcH5a, ubiquitin, and ATP. Lanes 4–7 contained Xnf7WT but lacked one component of the ubiquitylation reaction. Lane 4, −E1; lane 5, −UbcH5a; lane 6, −ATP; lane 7, −ubiquitin. Reactions were incubated at 37°C for 2 h and were analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin or anti-Xnf7 antibodies. White line indicates that intervening lanes have been spliced out. (C) Xnf7 can function with several E2s but cannot function with UbcH10. (left) Xnf7WT was incubated as in B but with each reaction containing only the E2 indicated. The absence of E2 in the reaction is indicated (−). (right) E2-thioester assays demonstrate that E2s used are functional. The presence of the E2 alone (−) and the E2 following a 30-min thioester assay (+) are indicated. Similar results were obtained for all E2s used (unpublished data). (D) Xnf7's E3 ligase activity is required for its effects on mitotic exit. CSF extracts were treated with buffer, Xnf7WT, or Xnf7m36 and incubated at 23°C for 20 min. After CaCl2 addition, aliquots were removed at the indicated times and analyzed by SDS-PAGE and immunoblotting with anti–cyclin B2 antibodies. (E) Xnf7m36 blocks the function of endogenous Xnf7 protein. CSF extracts were treated with buffer or increasing amounts of Xnf7m36 and incubated at 23°C for 20 min. After CaCl2 addition, aliquots were removed and analyzed as in D. (F) Xnf7's ubiquitin ligase activity is required for its effect on APC substrate ubiquitylation. Methyl-ubiquitin was added to the experiment described in D. Aliquots were removed at the indicated times, analyzed by SDS-PAGE and autoradiography, and quantitated by Phosphorimager. White lines indicate that samples run on the same gel were reordered. Image published in: Casaletto JB et al. (2005) Copyright © 2005, The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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