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. 2018 Nov 8;47(1):468–479. doi: 10.1093/nar/gky1108

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Validate the binding interface between CENP-H and CENP-I, and the assembly mode of CENP-H/I/K ternary complex. ( A) Zoomed-in view of the binding interface between thCENP-H^CT and ctCENP-I^NT. The interacting residues were drawn in stick and marked in number. (B) The relative binding activity of thCENP-H (WT and mutants) bound to GST-ctCENP-I^NT was assessed using GST pull-down assays. Error bars represent standard deviations, which were obtained from three independent experiments. The representative results of the pull-down assays were also available in Supplementary Figure S8A. (C) In vitro binding assays were performed to examine the binding activity among the ternary complex. Proteins with distinct combinations as indicated were mixed and GST pull-down assays were performed. The bead-bound proteins were resolved with SDS-PAGE and stained with Coomassie Blue. (D) In vitro binding assays were performed to assessing the effects of thCENP-H mutants on the formation of the fungal CENP-H/I/K ternary complex. Proteins with distinct combinations as indicated were mixed and GST pull-down assays were performed. The bead-bound proteins were resolved with SDS-PAGE and stained with Coomassie Blue. (E) Quantification of the relative binding activity between ctCENP-I^NT and thCENP-H (WT and mutants) in the presence of thCENP-K based on the results from lanes 5, 6 and 7 in (D). (F) Zoomed-in view of the binding interfaces of thCENP-H^CT in the complex structure. The interacting resides in thCENP-H were drawn in stick and marked with numbers in red. And the counterpart residues in hsCENP-H were shown with numbers in black. ( G) HeLa Tet-On cells transfected with GFP-hsCENP-H (WT or mutants), non-tagged hsCENP-K and MYC-hsCENP-I. Cell lysates were treated with anti-GFP antibody. Immunoprecipitated samples were resolved with SDS-PAGE and blotted with the indicated antibodies. ( H) Quantification of the relative binding activity between hsCENP-I and hsCENP-H WT or mutants (upper panel), and between hsCENP-K and hsCENP-H WT or mutants (lower panel) based on the results from Figure 4G.

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