ETAA1 regulates phosphoproteins at kinetochores and spindles. (A) Flag-ETAA1 was exogenously expressed in 293T cells, immunopurified, and copurifying proteins were identified by mass spectrometry. Shown are the peptide counts for two experiments with two negative controls and two Flag purifications from parental cells lacking Flag-ETAA1. (B) HCT116 cells with or without stable expression of Flag-ETAA1 were treated with 100 nM taxol for 2 h and fixed, and Flag/BUB1 PLA signals were measured in mitotic and interphase cells. Bar, 5 µm. Quantification is the mean and standard deviation of three independent experiments in which at least 100 cells were analyzed for each condition. Significance was determined by ANOVA with a Dunnett multiple comparison post-test. (C) Diagram depicting the kinetochore- and spindle-associated phosphoproteins and sites detected in the ETAA1-dependent phosphoproteome. Each large gray circle contains the protein name with the smaller surrounding circles containing the observed phosphosites. The inner and outer phosphosite circle colors indicates the quantitative dependency on ETAA1 and TOPBP1, respectively.