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. 2005 Apr 11;169(1):61–71. doi: 10.1083/jcb.200411056

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Copyright © 2005, The Rockefeller University Press

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Figure 1. — Immunodepletion of Xnf7 accelerates exit from CSF arrest. (A) Xnf7 interacts with the cyclin B2 CRS. Glutathione-Sepharose beads were coupled to GST, GST-cyclin B1 CRS, or GST-cyclin B2 CRS and incubated with egg extracts. (left) Beads were washed, and bound material was conjugated to biotin. Biotinylated material was resolved by SDS-PAGE, transferred to nitrocellulose, and probed with HRP-streptavidin. The left lane of each panel shows proteins present on the beads alone; the right lanes show proteins bound after incubation with extract. (right) Beads were washed and bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. The arrows indicate the ∼80-kD band that was identified by mass spectrometry as Xnf7. (B) Characterization of Xnf7 antibodies. Xenopus XTC cell lysate and interphase and CSF-arrested egg extracts were resolved by SDS-PAGE and immunoblotted with affinity-purified Xnf7 antibodies (left) or purified preimmune IgG (right). (C) Xnf7 wild-type (Xnf7WT) and Xnf7 mutant 36 (Xnf7m36) expressed in Escherichia coli, rabbit reticulocyte lysate (RRL) programmed with Xnf7 (Xnf7 TNT), and unprogrammed RRL were resolved by SDS-PAGE and immunoblotted with affinity-purified Xnf7 antibodies. (D) Immunodepletion of Xnf7 from egg extracts. Purified anti-Xnf7 antibodies or purified preimmune IgG were coupled to protein A–Sepharose and incubated with extracts. Three consecutive depletions were performed and depleted extracts were resolved by SDS-PAGE and immunoblotted with anti-Xnf7 antibodies. (E) Immunodepletion of Xnf7 accelerates the degradation of cyclin B1 and cyclin B2 during exit from CSF arrest. CSF extracts were depleted 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 by SDS-PAGE and immunoblotting with anti–cyclin B1 or anti–cyclin B2 antibodies. (F) Immunodepletion of Xnf7 accelerates the degradation of securin during exit from CSF arrest. CSF extracts were depleted, supplemented with ³⁵S-securin, and incubated at 23°C for 30 min. CaCl₂ was added to extracts, and aliquots removed at the indicated times were analyzed by SDS-PAGE and autoradiography.