M18BP1.S competes for HJURP binding to CENP-C. (a) Schematic of the domains of HJURP and M18BP1.S that compete for binding to the CENP-C C-terminus. (b–d) Coomassie stained gel of purified proteins used in c and d. Proteins are indicated on top and their full-length migration position to the right. The molecular weight standards in kD are indicated to the left. (c) Addition of M18BP1.S to undepleted or M18BP1-depleted metaphase extract competes for the interaction between HJURP^S220A or HJURP^P221A and CENP-C. HJURP mutants indicated across the top were added to metaphase X. laevis extract that had been depleted with M18BP1 antibody or mock-depleted with IgG (∆). Extract supplemented with the 161–580 fragment of M18BP1.S are indicated (+). The top panel contains 5% of the input material and the bottom panel contains the immunoprecipitates after CENP-C precipitation and Western blotting for CENP-C, FLAG (HJURP), and M18BP1. (d) The SANTA domain mutant of M18BP1.S that cannot bind CENP-C fails to compete for HJURP^S220A or HJURP^P221A binding. Extracts were manipulated as in c with the addition of two different concentrations of M18BP1.S^161-580 or the addition of the M18BP1.S^SANTA mutant as indicated on the left. The top panel contains 5% of the input material and the bottom panel contains the immunoprecipitates after CENP-C precipitation and Western blotting for CENP-C, FLAG (HJURP), and M18BP1. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG serve as negative controls. Source data are available for this figure: SourceData F5.