A fraction of Cdt1, Ska1, and Ndc80 form a tripartite complex that exhibit synergy in binding to microtubules in vitro. (A) HeLa cells stably expressing HA-tagged Cdt1-WT were immunoprecipitated (IP) using an anti-HA antibody and the Western blot was probed with the antibodies indicated on the right. (B) Elution profiles of indicated proteins/complexes or mixture of proteins/complexes from the co-fractionation experiments using Superose 6 gel filtration chromatography. Zoomed version from 1.5 to 2.8 ml is shown inset. (C) Western blots of fractions from B using primary antibodies as indicated on the right, for the proteins run separately or in combination as indicated on the left. The initial three elution fractions (19, 20, and 21) from a mixture of Ndc80+ Cdt1-WT + pSka1 complex have been marked with a red box to highlight the observed difference with those of the individually run proteins/complexes. (D) Selected images showing binding at single-molecule level of HEC1/NUF2-GFP, and SKA1/2^Cy3 on taxol stabilized Alexa 647-biotin-labeled microtubules both in the absence (left) and in the presence (right) of 1 nM untagged Cdt1. Colocalized bindings are indicated with white arrowheads. (E) Scatter plot of the fraction of colocalized binding sites at three different concentrations of untagged Cdt1. Bar and whiskers are mean ± SEM. *** P < 0.0001, ** P < 0.001 (Mann–Whitney U test). (F) Selected images showing binding at single molecule level of GFP-Cdt1, SKA1/2^Cy3, and Ndc80^alexa647 proteins on taxol-stabilized biotin-labeled microtubules (not imaged). Colocalized bindings are indicated with white arrowheads. (G₁and G₂) Two examples showing the binding of single molecules GFP-Cdt1 (i), SKA1/2/3^Cy3 (ii), and Ndc80^alexa647 (iii) proteins to Taxol-stabilized biotin-labeled microtubules. Colocalized bindings are indicated with white arrowheads. Source data are available for this figure: SourceData F7.