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Figure 1


Fig. 1. p97 is not essential for spindle disassembly. (A,B) CSF-extracts were immunodepleted with the antibodies anti-Ufd1, anti-Npl4, anti-Ufd1 and anti-Npl4, anti-p47 or unspecific IgG and immunoblotted after the first and second depletion. Equivalent amounts and one-tenth of untreated extracts served as controls. Asterisks denote unspecific bands. (C) CSF-extracts were supplemented with Xenopus, murine or human p97 isoforms (wild type or dominant-negative mutants at various final concentrations) or stable human cyclinB1. Alternatively, extracts were depleted ({Delta}) with the antibodies anti-Ufd1, anti-Npl4, anti-Ufd1 and anti-Npl4, or anti-p47 and, where indicated, supplemented with soluble anti-Ufd1 or anti-Npl4 antibodies. Sperm-induced spindles were assembled for 30 minutes at 20°C. After triggering release into interphase, Rhodamine-labelled MTs and DAPI-stained sperm chromatin were examined. The two micrographs are typical examples of spindles immediately before (– Ca2+) and 60 minutes after release (+ Ca2+). Bars, 10 µm. (D) Translation of CFTR{Delta}F508 mRNA in rabbit reticulocyte lysate or Xenopus low-speed extracts followed by CFTR western blotting. Arrowheads indicate CFTR-specific bands, all unlabeled bands are unspecific. (E) p97-QQ-supplemented versus buffer-supplemented extracts, incubated with CFTR{Delta}F508 mRNA for 3 hours before quantifying CFTR relative to beta-tubulin by western blotting. Shown is the mean (± s.d.) of three independent experiments. (F) Egg extract was incubated with human CFTR{Delta}F508 mRNA for 90 minutes at 20°C prior to addition of Xenopus p97-QQ (final 1 mg/ml) or reference buffer. Fifteen minutes later, translation was stopped with cycloheximide and degradation of CFTR{Delta}F508 was analyzed by western blotting. Lanes 7 and 14 show steady-state levels for one out of three experiments quantified in E. (G) ATPase assays of recombinant p97 proteins. The OD615 values at 0 minutes were substracted from OD615 values at 15 and 30 minutes.





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