SET binding to Sgo1 inhibits the Sgo1–cohesin interaction in cells and in vitro. (A) SET binding to Sgo1 inhibits the Sgo1–cohesin interaction in vitro. Recombinant SA2/Scc1 complexes (SA2: 80–1060; Scc1: 281–420) were incubated with GST-Sgo1 fragments (241–387) in the presence or absence Cdk1/cyclin B1. GST pull-down was performed in the presence of His₆-SET with increasing concentrations. Pelleted proteins were resolved with SDS-PAGE, stained with Coomassie blue, or blotted with antibody against Sgo1 pT346. (B) SET overexpression weakens the endogenous Sgo1–cohesin interaction. Lysates of nocodazole-arrested HeLa Tet-On cells transfected with vectors or plasmids containing GFP-SET were incubated with IgG or with antibody against Sgo1. The immunoprecipitated proteins were resolved with SDS-PAGE and blotted with the indicated antibodies. H denotes heavier exposure. (C) Quantification of Sgo1-bound Smc1 in B. A two-tailed t-test was performed for the experiments. Quantification details were recorded in the section of Antibodies, immunoblotting, and immunoprecipitation in Materials and methods. Averages and standard deviations were calculated based on two independent experiments. *, P < 0.05. (D) Overexpression of SET WT, not 3K, moderately weakens the Myc–Sgo1–cohesin interaction in cells. Lysates of nocodazole-arrested HeLa Tet-On cells stably expressing Myc-Sgo1 (1–472) transfected with vector controls (V) or plasmids containing GFP-SET WT or 3K were incubated with antibody against Myc. The immunoprecipitated proteins were resolved with SDS-PAGE and blotted with the indicated antibodies. (E) Quantification of Myc-Sgo1–bound Smc1 in D. One-way ANOVA was performed followed by pairwise comparisons using Tukey’s test. Quantification details were recorded in the section of Antibodies, immunoblotting, and immunoprecipitation in Materials and methods. Means and standard deviations were calculated based on three independent experiments. *, P < 0.05. n.s., not significant. (F) SET disrupts the Sgo1–cohesin binding in a dose-dependent manner. Lysates of nocodazole-arrested HeLa Tet-On cells stably expressing Myc-Sgo1 (1–472) transfected with vectors or different doses of plasmids containing GFP-SET were incubated with antibody against Myc-Sgo1. The immunoprecipitated proteins were resolved with SDS-PAGE and blotted with the indicated antibodies. (G) SET overexpression does not affect Sgo1 pT346 levels. Nocodazole-arrested HeLa Tet-On cells stably expressing Myc-Sgo1 (1–472) were transfected with vectors (Mock) or plasmids containing GFP-SET WT or 3K. Different doses of cell lysates were loaded, resolved on SDS-PAGE, and blotted with the indicated antibodies.