Figure 1.

Overview of the approach to generate core SAC protein BioID2-proximity association networks. (A) Schematic of the core spindle assembly checkpoint (SAC) components BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 that localize to the kinetochore region during early mitosis. MCC denotes a mitotic checkpoint complex. (B) Generation of inducible BioID2-tagged stable cell lines for each core SAC protein. (C) Fixed-cell immunofluorescence microscopy to analyze BioID2-tagged SAC protein subcellular localization in time and space. (D) Biochemical purifications; affinity purification of biotinylated proteins; and identification of proteins by liquid chromatography with tandem mass spectrometry (LC/MS/MS). (E) Computational analysis of raw mass spectrometry data using in-house R scripts. (F) Generation of high-confidence SAC protein proximity association networks.