![Figure 6. Recruitment of HJURP to centromeres requires the Mis18 complex. (A and B) Cellular extracts from siRNA-treated and control cell lines were analyzed by Western blotting using anti-GFP (A) or anti-HJURP antibodies (B). Each lane contains lysate from 105 cells. Dilution series were generated from mock-treated HeLa GFP-Mis18α (A) or parental HeLa (B) cells. (C) Stable GFP-Mis18α cells lines were treated with siRNA against Mis18α, Mis18BP1hsKNL2, HJURP, or GAPDH (control). Representative images of siRNA-treated GFP-Mis18α cells were selected in which a midbody was clearly present (differential interference contrast [DIC], arrows) to show the cell was in early G1. DAPI staining was overlaid onto the differential interference contrast image. Cells were stained with anti–CENP-T. (D) Mean percentage of GFP-Mis18α centromere-positive nuclei from a population of ≥57 cells in each siRNA treatment from two experiments. (E) Similar image acquisition as in C. Here, stable HeLa GFP-HJURP cells were treated with the same siRNAs. (F) Mean percentage of GFP-HJURP centromere-positive nuclei from a population of ≥135 cells in each siRNA treatment from two experiments. Error bars show standard deviations. Insets show magnified views of boxed regions. Bars, 5 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/194/2/10.1083_jcb.201012017/6/jcb_201012017r_rgb_fig6.jpeg?Expires=1771882135&Signature=m4xVH8ido8BWSWhcnzJ-wMzGVFwzMglo8yH8K6RXC7cydbBUXpCalA37I8lmsdvBEYtcny~yrGjMgLzSFT6jYwrGp29CXV9QWQ6YxVYCVJs-96lw1jjQFPtxEuBAJzcK8UG--8NvSw01nRJRgQo7kGIfeaRvmZ3HcJTC2BoTl7mPZz58QDpWC-5fxLChDW4V-VmqnfdfEJDlOOdWR3Ur3JunemDDo38UZXmxV7-JPRS-EU3TOjc51v3tNeIFXADgHblDaplGvR~Spu4jGe9dEpE8IObCzT5ubZxLhdrXxr~0L9eVU9gbhxo5XqBE9R09eLo1xTUjlhxuDQN7aKMFPw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Recruitment of HJURP to centromeres requires the Mis18 complex. (A and B) Cellular extracts from siRNA-treated and control cell lines were analyzed by Western blotting using anti-GFP (A) or anti-HJURP antibodies (B). Each lane contains lysate from 105 cells. Dilution series were generated from mock-treated HeLa GFP-Mis18α (A) or parental HeLa (B) cells. (C) Stable GFP-Mis18α cells lines were treated with siRNA against Mis18α, Mis18BP1hsKNL2, HJURP, or GAPDH (control). Representative images of siRNA-treated GFP-Mis18α cells were selected in which a midbody was clearly present (differential interference contrast [DIC], arrows) to show the cell was in early G1. DAPI staining was overlaid onto the differential interference contrast image. Cells were stained with anti–CENP-T. (D) Mean percentage of GFP-Mis18α centromere-positive nuclei from a population of ≥57 cells in each siRNA treatment from two experiments. (E) Similar image acquisition as in C. Here, stable HeLa GFP-HJURP cells were treated with the same siRNAs. (F) Mean percentage of GFP-HJURP centromere-positive nuclei from a population of ≥135 cells in each siRNA treatment from two experiments. Error bars show standard deviations. Insets show magnified views of boxed regions. Bars, 5 µm.
