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. 2003 Oct 1;17(19):2421–2435. doi: 10.1101/gad.1126303

Figure 4.

Figure 4.

KNL-1 forms a complex with two widely conserved outer kinetochore proteins. (A) Silver-stained gel of immunoprecipitates from embryo extracts. The three bands consistently detected in KNL-1 (α-KNL-1_NT2) but not control (Random IgG) immunoprecipitates (indicated by arrows; n = 4 extracts) were identified as KNL-1, CeNDC-80, and HIM-10 by mass spectrometry. The same bands were detected in immunoprecipitates using a second KNL-1 antibody (data not shown). Contaminating antibody heavy (H) and light (L) chains and background bands (*) are also indicated. (B) Western blots of embryo extracts probed with affinity-purified antibodies against CeNDC-80 and HIM-10. Asterisks indicate background bands that were not eliminated by RNAi of the corresponding genes. The molecular mass markers were 200, 97.4, 66.2, 45, 31, and 14.4 kD. (C) Western blots of KNL-1 immunoprecipitates probed with the indicated antibodies. Identical results were obtained in immunoprecipitations performed on four independent extracts. (D) Western blots of CeCENP-C immunoprecipitates probed with the indicated antibodies. Identical results were obtained in immunoprecipitations performed on two independent extracts. CeCENP-C was not detectable by silver staining in either KNL-1 or CeCENP-C immunoprecipitates. (E) 3D widefield data sets were acquired every 10 sec for wild-type (n = 27), KNL-1-depleted (n = 24), CeNDC-80-depleted (n = 21), and HIM-10-depleted (n = 22; not shown; see Supplementary Videos 11, 12) embryos. Time-aligned projections of representative 3D movies are shown (see also Supplementary Videos 7-14). The times after NEBD are indicated on the left in seconds. Bar, 5 μm. (F) The distance between spindle poles was tracked for 11 CeNDC-80-depleted embryos filmed as in E. The plot shows average pole-to-pole distance versus time after NEBD. Wild-type and KNL-1-depleted tracking data are replotted from Figure 1C. Error bars represent the S.E.M. with a confidence interval of 0.95. The average times of the onset of cytokinesis (all three conditions) and the average time when chromosome separation was first visible (wild-type and CeNDC-80-depleted embryos) are also indicated.