![Figure 4. In vivo analysis of CPC formation. (A and B) INCENPON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENPOFF cells expressing TrAP-INCENPWT (C and D), TrAP-INCENPW766G (E and F), or TrAP-INCENPF802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENPOFF/TrAP-INCENPW766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/187/5/10.1083_jcb.200906053/6/jcb_200906053_rgb_fig4.jpeg?Expires=1767751878&Signature=o4S7pojwo1~0vQNKY8lmEcF6bvKFPHtfqozVzwTu24AqfxpBv93L-4HIqUIrtixJj~n-QSTFUt67zaDzWQ0sovDOCmCpw7cMl5Xf6Re6BvROYutV4NGV2e-Fpk9WOnqdXX1D2pzlym9tTySOtoWCd8brl1C2jj0D7bPVyld3kpz5NfAv7K2TfP-nCwu0OSWrE84sEUEYuWZO~3f~naJn4zVKp-X6X46AEujbIYxgpDwQ3jFCkmOhsbFRVjE5joOSZBb~FEYtfnf5XQ~DKh4tGhaAM7gb5ZwXDtoNgQ1oV9R0EUfhCZhRF~5Iwv2QvdC~eG2UsdUsJ8R4OaVkSXTmGQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
In vivo analysis of CPC formation. (A and B) INCENPON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENPOFF cells expressing TrAP-INCENPWT (C and D), TrAP-INCENPW766G (E and F), or TrAP-INCENPF802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENPOFF/TrAP-INCENPW766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.
