The following antibodies were used in this study: anti-centromere antibody (ACA or CREST-ImmunoVision, HCT-0100), anti-PP2A-Aα (Santa Cruz, Sc-6112), anti-Histone H3-pS10 (Cell signaling, 9706), anti-Smc1 (Bethyl, A300-055A), anti-SET (Bethyl, A302-261A), AKT (Cell signaling, 4691S), pAKT (S473) (Cell signaling, 4060S), anti-actin (Thermo Scientific, MA5-11869), anti-pHec1 (phospho Ser55, GTX70017, GeneTex), and anti-Myc (Millipore, 11667149001). Anti-Sororin is a gift from Susannah Rankin. Anti-Sgo1 and anti-GFP antibodies were made in-house as described previously (Liu et al., 2013b (link); Kim and Yu, 2015 (link)).
Antibody dilution for immunoblotting was often 1:1000 unless specified.
The secondary antibodies were purchased from Li-COR: IRDye 680RD goat anti-mouse IgG secondary antibody (926-68070) and goat anti-rabbit IgG secondary antibody (926-32211).
Harvested cells were collected and lysed with SDS sample buffer. After being 5-min boiled, lysates were resolved by SDS–PAGE and blotted with indicated antibodies.
For immunoprecipitation, anti-Myc or anti-GFP antibodies were coupled to Affi-Prep Protein A beads (Bio-Rad) at a concentration of 1 mg/ml−1.
Antibody dilution for immunoblotting was often 1:1000 unless specified.
The secondary antibodies were purchased from Li-COR: IRDye 680RD goat anti-mouse IgG secondary antibody (926-68070) and goat anti-rabbit IgG secondary antibody (926-32211).
Harvested cells were collected and lysed with SDS sample buffer. After being 5-min boiled, lysates were resolved by SDS–PAGE and blotted with indicated antibodies.
For immunoprecipitation, anti-Myc or anti-GFP antibodies were coupled to Affi-Prep Protein A beads (Bio-Rad) at a concentration of 1 mg/ml−1.