Immunoprecipitation and competition assays

Inducible, stable cell lines expressing indicated YFP‐tagged bait were lysed in a low salt lysis buffer (50 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DDT, 0.1% NP‐40, protease‐ and phosphatase inhibitors), unless otherwise stated. In some experiments, the same lysis buffer with 150 mM NaCl was used. Lysates were immunoprecipitated with GFP‐trap beads (ChromoTek) according to the manufacturer’s recommendation. The beads were washed three times with wash buffer (50 mM Tris–HCl pH 7.5, 1 mg/ml BSA, 20% glycerol, and 1 mM DTT) and eluted in 2× sample buffer. For the peptide competition assays, a peptide containing LxxIxE motif (LPRSSTLPTIHEEEELSLC) or a control mutant peptide that was unable to bind B56 (LPRSSTLPTAHAEEELSLC) was used. For the competition assay with hSgo1 proteins, purified full‐length hSgo1 or hSgo1_1–155 described above were used. The peptides/proteins were incubated with cell lysates 30 min prior to the addition of GFP‐trap beads.

IP samples were resolved with 4–12% Bis‐Tris gels (Thermo Fischer Scientific) and transferred to PVDF membranes. LI‐COR Odyssey imaging system was used for visualization, and signals were quantified using Image Studio software (LI‐COR).