MAD1 is the kinetochore receptor for CCNB1. (A and B) CCNB1-mCherry (CCNB1-mCh) was immunoprecipitated (IP) with anti-mCherry antibodies from CCNB1-mCh or parental HeLa cells arrested in mitosis. Coprecipitating proteins were analyzed by mass spectrometry in three independent experiments (A) or by Western blotting (B). Intensities of proteins identified by mass spectrometry in all experiments were plotted against each other. Nonspecific components of the IPs are found equally in both samples and therefore align along the diagonal. Specific components of CCNB1 complexes cluster along the y-axis of the plot. (C) Cyclin IPs were performed from HEK293T cells cotransfected with full-length mCh-MAD1 and Myc-CCNA2, CCNB1 or CCNB2 constructs indicated. Myc IPs were Western blotted for Myc to detect precipitated cyclins, for CDK1, and for mCh to detect coprecipitated MAD1. Asterisks mark the antibody light chain detected in the CDK1 blots for IP samples. (D) Prometaphase localization of CCNB1 in control (siControl) and MAD1 (siMAD1) depleted HeLa CCNB1-GFP cells arrested with 20 µM MG132 for 30 min, then stained for MAD1 and kinetochores (CREST). Kinetochore fluorescence of MAD1 and CCNB1 normalized to the respective mean signal in control cells and SEM are plotted in the bar graphs (15 kinetochores per cell and 12 cells in each of three independent experiments). (E) Prometaphase localization of MPS1 in control (siControl) and MAD1 (siMAD1) depleted HeLa MPS1-mCherry cells stained for MAD1 and kinetochores (CREST). Kinetochore fluorescence of MAD1 and MPS1 normalized to the respective mean signal in control cells and SEM are plotted in the bar graphs (n = 19, P value for MPS1-mCherry < 0.0001, Student’s t test).