Analyses of binding between Cdt1, Ska1, and their fragments. (A) Pull down of the SKA1 and the SKA3 subunits of the Ska1 complex by HA/His-tagged Cdt1-WT from thymidine synchronized and nocodazole arrested mitotic HEK-293T cell extracts. The pull down was performed using Ni2+-NTA agarose beads followed by immunoblotting with either anti-HA or anti-SKA3 and SKA1 antibodies. 1% of the lysate was loaded as total protein. (B) Thymidine synchronized and nocodazole arrested mitotic HeLa cells stably expressing GFP were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies; IgG was taken as a negative control. 1% of the lysate was loaded as input. (C) SDS-PAG electrophoresis of purified His-SKA1/2 dimer. (D) Linear protein diagrams of the truncated protein constructs used in the BLI-interaction assay with indicated amino acids position showing truncation sites. (E) BLI sonograms for indicated conditions and their respective Kd values. (F) SDS-PAGE gel electrophoresis of purified SKA1/2+ SKA2-GFP complex. Source data are available for this figure: SourceData FS3.
