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. 2005 Apr 11;169(1):49–60. doi: 10.1083/jcb.200411118

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Copyright © 2005, The Rockefeller University Press

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Figure 1. — ZW10 and Zwint-1 reside in distinct kinetochore subcomplexes in HeLa cells. (A) Localization of ZW10^LAPtag in a HeLa cell line stably expressing the fusion protein (clone LZ5). Cells were treated with nocodazole for 30 min before fixation, and stained for ZW10^LAPtag (anti-GFP), centromeres (ACA), and DNA (DAPI). (B) Localization of Zwint-1^LAPtag in clone LINT2.8. Treatment and staining was performed as in A, except cells were preextracted before fixation. (C) Immunoblot of whole cell lysates of HeLa cells and clone LZ5. Lysates were probed for ZW10, ZW10^LAPtag (anti-GFP), and tubulin. (D and E) Tandem affinity purification of Zwint-1^LAPtag (D) and ZW10^LAPtag (E) from mitotically arrested cells. 25% of eluate was analyzed by SDS-PAGE followed by silverstain and 75% was analyzed by MudPIT mass spectrometry. Name, percent sequence coverage, and expected molecular weight of the identified proteins are indicated in the table. Suspected position of the identified proteins on the silver stained gel are indicated on the right. Unlabeled bands on silverstain likely include DC31 (∼32 kD), Q9H410 (∼40 kD), and the nonspecific proteins HSP70 (∼70 kD) and α-tubulin (∼50 kD).