Figure 5.
Figure 5. Epsin1 affects spindle assembly in Xenopus egg extracts. (A) Characterization of XEpsin1 antibody. Rabbit polyclonal antibodies against the ENTH domain of XEpsin1 recognized a single band in Xenopus egg extract (XEE) by Western blot analysis. 6His-tagged XEpsin1 purified from bacteria showed an apparent smaller molecular mass than the endogenous protein. After incubating with the Xenopus egg extract that was immunodepleted of endogenous XEpsin1, the recombinant XEpsin1 migrated as a similar size as the endogenous protein (XEpsin1-6His in XEE). (B) XEpsin1 antibody inhibits spindle assembly in cycled Xenopus egg extracts. Quantifications of percentages of MT structures formed in cycled extract after the addition of control antibody (+control rabbit IgG) or XEpsin1 antibody (+anti-XEpsin1) were performed by analyzing structures associated with >300 sperm in each reaction. Results from four independent experiments were averaged. (C) Examples of Xenopus sperm associated with normal spindle, abnormal MT structures, or no MT structures. MT structures were visualized by rhodamine-tubulin, and chromosomes were stained with DAPI. (D) Western blot analysis of XEpsin1 in extracts after immunodepletion. Different amounts of untreated egg extracts were loaded as standards. Mock-depleted extract (−IgG) had a similar level of XEpsin1 as nondepleted extract, whereas ∼90% of XEpsin1 was depleted by XEpsin1 antibody (−XEpsin1). (E) Western blot analysis of XEpsin1 after depletion (−XEpsin1) and add back of recombinant wild-type or mutant XEpsin1 (−XEpsin1 + WT or −XEpsin1 + L6E) compared with mock-depleted extract. (F) Coomassie blue staining of SDS-PAGE to show the quality of purified wild-type (WT) and mutant (L6E) XEpsin1 proteins. (G) Effects of depletion/add back of XEpsin1 on spindle assembly in CSF extracts. Mock-depleted extract, XEpsin1-depleted extract (−XEpsin1), or XEpsin1-depleted extract with the addition of wild type or the L6E mutant of the XEpsin1-6His protein was allowed to form spindles by incubation with sperm DNA. The number of normal spindles was quantified and normalized against mock-depleted extract. Results from three independent experiments, each containing structures associated with >300 sperms per reaction, were averaged. *, P < 0.05; **, P < 0.01. (B and G) Error bars show standard deviation. Bar, 10 μm.

Epsin1 affects spindle assembly in Xenopus egg extracts. (A) Characterization of XEpsin1 antibody. Rabbit polyclonal antibodies against the ENTH domain of XEpsin1 recognized a single band in Xenopus egg extract (XEE) by Western blot analysis. 6His-tagged XEpsin1 purified from bacteria showed an apparent smaller molecular mass than the endogenous protein. After incubating with the Xenopus egg extract that was immunodepleted of endogenous XEpsin1, the recombinant XEpsin1 migrated as a similar size as the endogenous protein (XEpsin1-6His in XEE). (B) XEpsin1 antibody inhibits spindle assembly in cycled Xenopus egg extracts. Quantifications of percentages of MT structures formed in cycled extract after the addition of control antibody (+control rabbit IgG) or XEpsin1 antibody (+anti-XEpsin1) were performed by analyzing structures associated with >300 sperm in each reaction. Results from four independent experiments were averaged. (C) Examples of Xenopus sperm associated with normal spindle, abnormal MT structures, or no MT structures. MT structures were visualized by rhodamine-tubulin, and chromosomes were stained with DAPI. (D) Western blot analysis of XEpsin1 in extracts after immunodepletion. Different amounts of untreated egg extracts were loaded as standards. Mock-depleted extract (−IgG) had a similar level of XEpsin1 as nondepleted extract, whereas ∼90% of XEpsin1 was depleted by XEpsin1 antibody (−XEpsin1). (E) Western blot analysis of XEpsin1 after depletion (−XEpsin1) and add back of recombinant wild-type or mutant XEpsin1 (−XEpsin1 + WT or −XEpsin1 + L6E) compared with mock-depleted extract. (F) Coomassie blue staining of SDS-PAGE to show the quality of purified wild-type (WT) and mutant (L6E) XEpsin1 proteins. (G) Effects of depletion/add back of XEpsin1 on spindle assembly in CSF extracts. Mock-depleted extract, XEpsin1-depleted extract (−XEpsin1), or XEpsin1-depleted extract with the addition of wild type or the L6E mutant of the XEpsin1-6His protein was allowed to form spindles by incubation with sperm DNA. The number of normal spindles was quantified and normalized against mock-depleted extract. Results from three independent experiments, each containing structures associated with >300 sperms per reaction, were averaged. *, P < 0.05; **, P < 0.01. (B and G) Error bars show standard deviation. Bar, 10 μm.

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