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. Author manuscript; available in PMC: 2016 Dec 17.
Published in final edited form as: Mol Cell. 2015 Nov 19;60(6):886–898. doi: 10.1016/j.molcel.2015.10.027

Figure 5. The CENP-T-W complex requires interactions with CENP-H-I-K-M and DNA for its centromeric localization.

Figure 5

A) SDS-PAGE gel showing co-purification of His-CENP-T-W and GST-CENP-I-H-K-M. CENP-W was not retained on the gel due to its small size. B) SDS-PAGE gel showing co-purification of His-CENP-T-W with untagged CENP-H-K, and GST-I-H-K-M as a positive control. C) SDS-PAGE gel indicating that His-CENP-T-W does not interact with GST-CENP-C. The sequential purification confirms that the bands co-purifying with His-CENP-T are contaminants and not GST-CENP-C. A parallel glutathione purification was performed (far right lane) to confirm the presence of GST-CENP-C in the extract. D) SDS-PAGE gel indicating the absence of an interaction of GST-CENP-T-W with CENP-L-N-His. E) Localization of transiently transfected GFP-CENP-W constructs in interphase cells. CENP-WDNA contains 5 mutations to disrupt its DNA binding (Nishino et al., 2012). Numbers represent percent of GFP-positive transfected cells showing the indicated phenotype, n = 100 cells. F) SDS-PAGE gel showing co-purification of GST-CENP-I-H-K-M with both His-CENP-T-W and His-CENP-T-WDNA. F) Schematic of the interactions underlying CENP-T-W localization. Gels stained were with Coomassie Brilliant Blue unless otherwise indicated. *: Contaminant. Scale bar, 5 μm.