Gfp trap beads

S2R + Drosophila cells were cultured in Schneider medium supplemented with 10% FCS, 10 U/ml penicillin and 10 µg/ml streptomycin. For plasmid transfections, 10.10⁶ cells per ml were seeded in 100 mm dishes and transfected with Effectene (Qiagen) according to the manufacturer’s protocol. Cells were harvested in Phosphate Buffered Saline (PBS) and pellets were resuspended in NP40 buffer (20 mM Tris pH7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 1 mM of DTT. For co-immunoprecipitation assays 1 mg to 1.5 mg of proteins were incubated for 3 hr with 15 µl of GFP-Trap beads (Chromotek). Beads were then washed 3 times with NP40 buffer and finally resuspended in Laemmli buffer for immunoblotting analysis. Input fractions represent 2.5% of the immunoprecipitated fraction.

HEK293T cells were plated at 1 × 10⁶ per 10-cm dish and transfected using standard calcium phosphate precipitation. Cells were lysed in buffer containing 50 mM Tris (pH 8), 0.5% Triton X, 40 mM NaCl, 2 mM CaCl₂, 20 mM nethylmaleimide, 1× protease inhibitor (Roche), 1× phosphatase inhibitor (Roche), and 25 U/mL Basemuncher (Expedeon). Lysates were kept on ice for 20 min before clearing by centrifugation at 13,500 rpm at 4°C for 15 min. Input samples were kept at −20°C. Lysates were then adjusted to 150 mM NaCl; anti-Myc antibody was added (final concentration ∼2 μg/mL); and samples were left rotating at 4°C for 1 hr. Protein G beads or GFP-Trap beads (chromotek) were washed three times in wash buffer (20 mM Tris [pH 8], 150 mM NaCl, and 20 mM nethylmaleimide) before the addition of lysate. Samples were then rotated overnight at 4°C. Beads were washed three times with wash buffer before resuspension in SDS loading buffer for analysis by immunoblotting. If Ni-NTA (Expedeon) beads were used for immunoprecipitation, lysates were adjusted to 150 mM NaCl and 30 mM imidazole. Wash buffer was also adjusted to 30 mM imidazole, while wash buffer containing 300 mM imidazole was used to elute bound proteins.

For Cdc20 and BubR1 immunoprecipitation, 2 μg of plasmid was transfected into HeLa cells in a 15 cm dish 48 h before collection. Cells were synchronized by double thymidine arrest followed by overnight treatment by nocodazole (200 ng/ml). Mitotic cells were shaken off the plate and lysed on ice in a lysis buffer containing 10 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP40 with protease and phosphatase inhibitors (Roche). The cell lysate was centrifuged at 17,000 g for 10 min at 4 °C, and the supernatant was applied to 20 μl of GFP-Trap beads (Chromotek) and shaken for 2 h at 4 °C. The beads were washed three times with 0.5 ml lysis buffer and boiled in 50 μl 2× SDS loading buffer. For Cdc20 knocking out examination, cells from a 10 cm dish were collected and lysed in 200 μl of lysis buffer as described above. The cell lysate was cleaned by centrifugation and boiled in an SDS loading buffer. A quantitative western blot (Odyssey DLx, LI-COR) was performed to examine Cdc20 and interested proteins. Antibodies used include Cdc20 (Santa Cruz, sc-13162; Bethyl, A301-180A), BubR1 (homemade in JN lab), Mad1 (Sigma, M8069), Mad2 (homemade in JN lab), Apc7 (Santa Cruz, sc-365649), Apc15 (Santa Cruz, sc-398488) and GAPDH (Proteintech, 60004-1). Fluorophore-labeled secondary antibodies include goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680CW (LI-COR).

HeLa cells transfected with securin FL, securin FF-A or empty mVenus N1 transfection vector were synchronised and collected as described above. Cells were then lysed in 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.5% NP-40 plus protease inhibitor cocktail (Roche) for 30 min on ice and clarified by a 12,000 × g spin for 20 min at 4°C. Complexes were immunoprecipitated for 90 minutes at 4°C with GFP-Trap beads (Chromotek). After five washes in lysis buffer, proteins were eluted from beads by incubating for 10 minutes at 95 °C in sample buffer. The supernatant was then analysed by immunoblotting.