SET binds to a domain in Sgo1 that is close proximity to the cohesin-binding domain, and Sgo1 ΔSET mutants fully support centromeric cohesion. (A) Myc-Sgo1 binds both isoforms of endogenous SET. Lysates of nocodazole-arrested HeLa Tet-On cells stably expressing Myc-Sgo1 were incubated with antibody against Myc. Pelleted proteins were resolved on SDS-PAGE and blotted with the indicated antibodies. IP, immunoprecipitation. (B) Mapping the domains in Sgo1 interacting with SET. GST proteins and GST-tagged Sgo1 fragments with the indicated lengths were incubated with His₆-SET (full length) proteins, and GST pull-down assays were performed. The pelleted proteins were resolved on SDS-PAGE and stained with Coomassie blue. (C) Schematic drawing of the functional domains in human Sgo1. (D) Recombinant GST-Sgo1 fragments were incubated with a cohesin sub-complex containing SA2 and Scc1 (left panel), or His₆-SET full length (right panel). The GST bead–bound proteins were resolved with SDS-PAGE and stained with Coomassie blue. SA2: 80–1060; Scc1: 281–420; GST-Sgo1 WT: 241–387; ΔSET: 241–387 depleted of 281–310; and ΔCohesin: 241–387 depleted of 313–353. Notably, Scc1 fragments are invisible in the gel stained with Coomassie blue, likely due to small size and small amount. Therefore, it was not labeled. The gels were spliced from the same ones. (E) The SET-binding domain in Sgo1 is required for the Sgo1–SET binding in cells. Lysates of mitotic HeLa Tet-On cells transfected with vectors (V) or plasmids containing GFP-SET and Myc-Sgo1 WT or ΔSET or ΔSET ΔN were incubated with antibody against GFP. The immunoprecipitated proteins were resolved with SDS-PAGE and blotted with the indicated antibodies. WT, Sgo1 full length; ΔSET, Sgo1 truncated of the region 281–310; ΔSET ΔN, Sgo1 truncated of the regions 1–40 and 281–310. The loading control is defined with a nonspecific Western blot band that appears in each lane. (F) The N-terminus (1–40) of Sgo1 is dispensable for the Sgo1–SET binding in cells. Lysates of nocodazole-arrested HeLa Tet-On cells transfected with vectors (V), plasmids containing GFP-SET and Myc-Sgo1 WT or ΔN were incubated with antibody against Myc. The immunoprecipitated samples were resolved with SDS-PAGE and blotted with the indicated antibodies. (G) The SET-binding domain and the N-terminus (1–40) in Sgo1 are dispensable for the Sgo1–cohesin binding in cells. Lysates of nocodazole-arrested HeLa Tet-On cells transfected with vectors (V) or plasmids containing Myc-Sgo1 WT, ΔSET, ΔN, or ΔN ΔSET were incubated with antibody against Myc. The immunoprecipitated proteins were resolved with SDS-PAGE and blotted with the indicated antibodies. (H) RPE-1 cells stably expressing exogenous Sgo1 WT or ΔSET were treated with Sgo1 siRNAs. Representative images of chromosome spread from nocodazole (3 h)–arrested RPE-1 cells are shown here. The outlined regions are amplified and shown in the right panel. (I) Quantification of cells with unseparated and separated sister chromatids in H. Unseparated sister chromatids were defined as the chromosome morphology described in Mock (H), and separated sister chromatids were defined as the chromosome morphology described in siSgo1 (H). Averages and standard deviations from two independent experiments are shown here. More than 30 cells for each condition were examined. (J) Cell lysates in H were separated with SDS-PAGE and blotted with the indicated antibodies.