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. 2008 Mar;19(3):843–854. doi: 10.1091/mbc.E07-06-0556

Figure 2.

Figure 2.

Role of CENP-O class proteins in kinetochore assembly. (A) Immunofluorescence analysis of WT and CENP-50–, -O–, -P–, -Q–, or -R–deficient cells. Cells were stained with anti-CENP-50, anti-CENP-O, anti-CENP-P, anti- CENP-Q, or anti-CENP-R antibody. Antibody signals were detected with FITC-conjugated secondary antibody (green). DNA was counterstained with DAPI (blue). Typical interphase (I) and mitotic (M) figures are shown. (B) Immunofluorescence analysis of CENP-H–conditional knockout (KO) cells (5-5). Cells were stained with anti-CENP-Q or anti-CENP-R antibody (green). +tet indicates addition of tetracycline to the culture medium. DNA was counterstained with DAPI (blue). (C) Immunofluorescence analysis of WT, CENP-Q-KO, or CENP-R-KO cells. Cells were stained with anti-CENP-H or anti-CENP-I antibody (green). DNA was counterstained with DAPI (blue). (D) Western blot analysis of CENP-50–, -O–, -P–, -Q–, or -R–deficient cells with anti-CENP-50, anti-CENP-O, anti-CENP-P, anti-CENP-Q, anti-CENP-R, or anti-CENP-H antibody. The amount of CENP-50 is decreased in CENP-Q–deficient cells, and the amount of CENP-P is decreased in CENP-O–deficient cells. The amount of CENP-R is decreased in all mutants, and CENP-H is stable in all mutants. A Coomassie Brilliant Blue (CBB)-stained gel is also shown. (E) Model of the association of CENP-O class proteins. CENP-O, -P, -Q, and -50 form a stable complex. In particular, CENP-50 is close to CENP-Q, and CENP-O is close to CENP-P. This stable complex functions downstream of CENP-H class proteins and upstream of CENP-R.