Kif23

The following primary antibodies were used: Actin: sc-47778 (Santa Cruz Biotechnology). KIF4A: A301-074A (Bethyl Laboratories). PRC1: sc-8356 (Santa Cruz Biotechnology). KIF14: A300-912A (Bethyl Laboratories). KIF20A: A300-879A/Bethyl Laboratories). KIF23: sc-867 (Santa Cruz Biotechnology). KIF2C: kind gift from Linda Wordeman. KIFC1: 12313 (Cell Signalling). ɑ-tubulin (Sigma T6074).

MGC-803 or HEK293T cells were transfected with or without siRNA or plasmid for 48 hours, and 100 μg of protein extract was diluted to1 ml in Co-IP buffer (Thermo, USA); then, 2 μg of KIF23 (Santa Cruz Biotechnology, USA) or FLAG (Cell Signaling Technology, USA) antibody was added to the protein samples, and the mixtures were incubated overnight at 4°C with rotation. Twenty microliters of 50% protein A/G-agarose bead slurry (equilibrated in Co-IP buffer) was added, and after 2 h of incubation at 4°C, the beads were washed 4 times with Co-IP buffer (1 ml per wash) and diluted with 1×loading buffer. Western blotting was performed, and β-catenin (Santa Cruz Biotechnology, USA), Amer1 (Abcam, USA), and APC (Cell Signaling Technology, USA) were detected.