Zwint1

The following antibodies were commercially purchased and used at the indicated dilutions for immunofluorescence (IF) and immunoblotting (IB); Actin (I-19, Santa Cruz Biotechnology, IB; 1/2000), α-tubulin (B-5-1-2, Sigma, IF; 1/1000), BubR1 (Bethyl Laboratories, IF; 1/500), CENP-E (Sigma, IF; 1/500, IB; 1/2000), CENP-F (Abcam, IF; 1/1000), CLIP170 (Santa Cruz Biotechnology, IF; 1/500), dynein intermediate chain (DIC, Sigma, IF; 1/500), Hec1 (9G3, Abcam, IF; 1/1000, IB; 1/2000), Kid (Cytoskeleton Inc., IB: 1/1000), KNL1 (Novus Biologicals, IF; 1/500), Mad2 (Novus Biologicals, IF; 1/500), Nde1 (Protein Tech Group, IF; 1/500), Ska1 (Abcam, IF; 1/500), Spindly (Bethyl Laboratories, IF; 1/500, IB; 1/2000), Zwint-1 (Bethyl Laboratories, IF; 1/500), Zw10 (Cosmo Bio, IF; 1/200, IB; 1/2000), pericentrin (Abcam, IF; 1/500).

Total proteins were extracted from samples using a mammalian cell extraction kit (BioVision, Inc., CA, USA). Equal amounts of protein were resolved using SDS–PAGE (12.5%), blotted onto a nitrocellulose membrane and then blocked in SuperBlock T-20 (PBS; ThermoFisher Scientific). The blots were incubated overnight at 4 °C with primary antibodies. The primary antibodies of cleaved caspase-3 (Cell Signaling Technology, Inc., MA, USA, #9661), cleaved poly-ADP-ribose polymerase (PARP) (Cell Signaling Technology, #5625), hnRNPA0 (Novus Biologicals, LLC., CO, USA, NBP2-22293), phosphor-hnRNPA0 (Signalway Antibody, LLC., MD, USA, 12686), ZWINT1 (Bethyl Laboratories, Inc., TX, USA, A300-781A), RAB3GAP1 (Proteintech Group, Inc., IL, Japan, 21663-1-AP), securin (Abcam, CB, United Kingdom, ab79546), Cyclin B1 (Abcam, ab32053), OPN3 (Abcam, ab75285), NUDT12 (Proteintech Group, Inc., 17487-1-AP) were diluted to 1:1000 in SuperBlock T-20 (PBS) and incubated with blots overnight at 4 °C. The blots were washed in T-PBS, incubated with HRP-conjugated secondary antibodies (R&D Systems, Inc., MN, USA), washed in T-PBS, and then developed using the Super-Signal West Pico enhanced chemiluminescence system (ThermoFisher Scientific). The averaged protein expression was normalized to the actin expression (BD Transduction Laboratories, KY, USA).

The cells were plated on chamber slides, which were fixed in 4% paraformaldehyde, washed extensively with PBS, permeabilized with 0.1% Triton X-100 and blocked in 3% BSA in PBS. The slides were then sequentially incubated with primary antibodies (α-tubulin [Novus Biologicals], active Rab3 [NewEast Bioscience, PA, USA], ZWINT1 [Bethyl Laboratories]) and washed with PBS and incubated with Alexa 594 or 488-conjugated secondary antibodies (ThermoFisher Scientific). The nuclei were counterstained with Hoechst 33342 (Invitrogen-Molecular Probes). The cells were mounted with an anti-fade mounting medium, and the immunofluorescence was visualized using a fluorescence microscope (KEYENCE Corporation).