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Files in this Data Supplement:
Fig. S1. (A) Immunoblots show FCPT (compared with DMSO for a control) did not enhance the binding of either TPX2 or γ-tubulin onto pelleted taxol polymerized microtubules in clarified Xenopus egg extracts. (Tubulin is a loading control.) (B) Immunoblots show GSK246053 (compared with DMSO for a control) enhanced the binding of kinesin-5 (approximately 3-fold), but not either XCTK2 or MCAK onto pelleted taxol polymerized microtubules in clarified Xenopus egg extracts. (Tubulin is a loading control.) (C) Similar to FCPT-treated spindles, GSK246053 treated (200 µM; GSK) Xenopus egg extract spindles had lower microtubule fluorescence at spindle poles compared with control spindles (Control). (Scale bar: 10 µm.) (D) Similar to FCPT, GSK246053 (200 µM) inhibits kinesin-5 motility on Xenopus egg extracts spindles. The kymographs (measured from red dotted line) of image sequences obtained over the course of approximately 1.5 minutes (with 1 image every 5 seconds) show that kinesin-5 is not motile when GSK246053 (200 µM) is present (GSK; shown as straight lines) while kinesin-5 is dynamic in untreated control spindles (Control). (Scale bar: 5 µm.)
Fig. S2. (A) Fixed images of FCPT-treated DMSO asters after (FCPT post-assembly) and before (FCPT pre-assembly) aster assembly − tubulin (red) and NuMA (green). FCPT addition after DMSO aster formation (FCPT post-assembly) had no effect on the localization of NuMA, while FCPT treatment before aster formation (FCPT pre-assembly) perturbed aster pole assembly and the pole localization of NuMA. (Scale bar: 10 µm.) (B) Fixed images of FCPT-treated RanGTP asters after (FCPT post-assembly) and before (FCPT pre-assembly) aster assembly − tubulin (red) and NuMA (green). Similar to DMSO asters, FCPT addition after RanGTP aster formation (FCPT post-assembly) had no effect on the localization of NuMA, while FCPT treatment before aster formation (FCPT pre-assembly) perturbed aster pole assembly and the pole localization of NuMA.
Movie 1. EB1-A488 real-time imaging of a control spindle. A real-time spinning-disc confocal image sequence of a Xenopus egg extract spindle supplemented with EB1-A488 (50 nM) imaged over the course of approximately 3.5 minutes (with 1 image ever 3 seconds). Movie is shown 10 frames per second.
Movie 2. EB1-A488 imaging of an FCPT-treated spindle. A real-time spinning-disc confocal image sequence of a Xenopus egg extract spindle supplemented with EB1-A488 (50 nM) and 200 µM FCPT imaged over the course of approximately 3.5 minutes (with 1 image ever 3 seconds). Movie is shown 10 frames per second.
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