Ybp2 physically associates with the COMA and Ndc80 complexes and Cse4.

(A–E) The indicated strains were grown to log phase at 30°C, lysed, and anti-myc immunoprecipitations were performed. Total lysate (T), supernatant (S), and the immunoprecipitated fraction (IP) were subjected to SDS-PAGE, and Western blots were used to detect Ybp2, Ctf19, Cse4, or myc-tagged proteins with the respective antibodies. For the quantification of Ybp2, aliquots of the total lysate (1/500 of the total), supernatant (1/500 of the total), and IP fraction (2/5 of the total lysate) were loaded. Arbitrary number is defined as the ratio of the amount of coprecipitated protein to the amount of input protein. Intensity of the total lysate is defined as 1 in each strain. Error bars indicate standard deviations of 2 or 3 experiments. The yeast strains used were (A) untagged (YPH499), Ybp2-myc (Y1689), Okp1-myc (Y1707), Mcm21-myc (Y1708), and Ame1-myc (Y1706); (B) untagged (YPH499), Ndc80-myc (Y1713), Nuf2-myc (Y1714), Spc24-myc (Y1715), and Spc25-myc (Y1716); (C) untagged (YPH499), Mtw1-myc (Y1709), Nsl1-myc (Y1710), Nnf1-myc (Y1711), and Dsn1-myc (Y1712); (D) Mcm16-myc (YVM325), Mcm22-myc (YVM290), Ctf3-myc (YVM219), Spc105-myc (Y1717), and Chl4-myc (YPH1542); and (E) untagged (YPH499) and Ybp2-myc (Y1689). Asterisks indicate IgG bands in immunoprecipitates. (F) Summary of coimmunoprecipitation assay results. Several kinetochore proteins were detected (red) in the Ybp2 immune complexes and others were not (brown).