Xenbase Image ID: 118896

Figure 3. Xnf7 modulates mitotic exit by altering ubiquitylation of APC substrates. (A) Xenopus egg extracts were depleted, supplemented with 35S-IAP, and treated with reaper and the broad-spectrum caspase inhibitor zVAD-fmk. Extracts were incubated at 23°C, and aliquots removed at the indicated times were analyzed by SDS-PAGE and autoradiography and quantitated by Phosphorimager. (B) Addition of anti-Xnf7 antibodies mimics immunodepletion of Xnf7. CSF extracts were treated with purified preimmune IgG, purified anti-Xnf7 antibodies, or purified anti-Xnf7 antibodies blocked with a COOH-terminal fragment of Xnf7 and incubated at 23°C for 15 min. After CaCl2 addition, aliquots were removed at the indicated times and analyzed by SDS-PAGE and immunoblotting with anti–cyclin B2 antibodies (left) or anti-Xnf7 antibodies (right). (C) Addition of recombinant Xnf7 to Xnf7-depleted extracts restores the normal timing of cyclin degradation. CSF extracts were depleted, treated with buffer or recombinant Xnf7, and incubated at 23°C for 30 min. CaCl2 was added to extracts, and aliquots removed at the indicated times after CaCl2 addition were analyzed as in B. (D) Addition of excess Xnf7 delays cyclin B degradation and mitotic exit. CSF extracts were treated with buffer or Xnf7WT and incubated at 23°C for 20 min. After CaCl2 addition, aliquots were removed at the indicated times and analyzed as in B. (E) Excess Xnf7 delays cyclin B 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. 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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