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. 2013 Apr 15;201(2):217–231. doi: 10.1083/jcb.201210033

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© 2013 Nijenhuis et al.

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Figure 2. — MPS1 kinetochore localization is mediated by the NTE-TPR module. (a) Schematic representation of the domain organization of various MPS1 proteins used throughout this study. (b) Immunoblot of whole-cell lysates from mitotic HeLa Flp-in LAP-MPS1 cell lines that were transfected with mock or MPS1 siRNA and induced (+ doxycycline) to express the indicated LAP-MPS1 proteins; band intensity of MPS1/tubulin relative to mock is indicated. (c) Immunolocalization of LAP-MPS1^1–192 and centromeres (CREST) in nocodazole-treated, MPS1-depleted HeLaK FRT TetR cells. Cells were imaged for prophase figures. DNA (DAPI) is shown in blue. Insets show magnification of the boxed regions. (d and e) Representative images (d) and quantification (e) of immunolocalization of the various LAP-MPS1 proteins and centromeres (CENP-A) in nocodazole, 500 nM reversine, and MG132-treated, MPS1-depleted Flp-in HeLa cells. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in e displays total kinetochore intensities (±SD) of the indicated LAP-MPS1 proteins relative to centromeres (CENP-A) in cells treated as in d. Data are representative of three experiments. Ratios for LAP-MPS1^WT are set to 1. One dot represents one cell. Line indicates means ± SD. ***, P < 0.0001; significant (Student’s t test, unpaired). Bars, 5 µm. WT, wild type; Tub, tubulin.