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ECFP (Clonetech) and EYFP (Clonetech) coding sequences were inserted into the pFastBac-p60 expression plasmid as in (15).
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21
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0344130087
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personal communication
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R. Tsien, personal communication.
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Tsien, R.1
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0344992630
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note
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Amino acid residues are abbreviated as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
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23
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0024520745
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note
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The E334Q mutant was prepared by overlap extension amplification [S. N. Ho et al., Gene 77, 51 (1989)] of the wild-type p60 cDNA, followed by cloning of the mutant sequence into pFastBac HT B as in (15) and sequencing to check for amplification errors.
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0014007813
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note
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2, 0.02% Triton X-100, 5% glycerol, and 1 mM ATP. Standard proteins were BSA (Stokes radius 35 Å), catalase (52 Å), and thyroglobulin (86 Å). We estimated molecular mass by using the measured Stokes radius and the S value [L M. Siegel and K. J. Monty, Biochim. Biophys. Acta 112, 346 (1966)].
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27
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0345423744
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note
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em 508).
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28
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0344130086
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note
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2-terminus of GFP.
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31
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0345423742
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note
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We performed ATPase assays by using the malachite green method as described in (7). We included DEAE-purified ATP at 1 mM.
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32
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0344130085
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We also examined FRET when the katanin concentration was varied with a constant microtubule concentration in the presence of ATP-γ-S. The signal did not increase in a hyperbolic manner. Instead, an increase in the FRET signal was observed only when the katanin reached a critical concentration, which again suggests a cooperative phenomenon.
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33
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0344561640
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c2+ depolymerized)]/microtubule concentration, μM).
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37
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0033534575
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39
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0344561639
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note
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We thank F. J. McNally, R. D. Mullins, K. S. Thorn, and J. E. Wilhelm for critical reading of the manuscript.
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