BRISC associates with the SAF NuMA during mitosis. (A) The ectopic FH–ABRO1 complex interacts with endogenous NuMA. Immunoprecipitate assays were performed on mock or FH–ABRO1 stably expressing HeLa cells with anti–FLAG M2 agarose gel, and analyzed by immunoblotting with the indicated antibodies. (B) The endogenous ABRO1 interacts with endogenous NuMA during mitosis. Immunoprecipitate assays were performed with anti-ABRO1 antibody in HeLa cells synchronized by double thymidine block or a sequential thymidine/NOC block release protocol. Immunoprecipitates were detected by immunoblotting with the indicated antibodies. For ABRO1 detection, a secondary antibody (A25022; Abbkine) specific against the light chain of IgG was used to exclude the interference of IgG heavy chain. Cyclin B1 was used as a G2/M index. (C) NuMA directly binds to the BRISC complex in vitro. GST–MERIT40 and GST–ABRO1/BRCC36 fusion proteins were purified from bacterial or insect cells. GST pull-down assays were performed using the purified GST fusion proteins and HeLa mitotic cell lysate. The bound protein complexes were analyzed by SDS-PAGE and visualized by Ponceau staining (bottom) and immunoblotting with an anti-NuMA antibody. (D) ABRO1 colocalizes with eYFP-NuMA at spindle poles. eYFP-NuMA stably expressing HeLa cell lines were fixed by cold methanol and stained with anti-ABRO1 antibody (red). DNA was stained with DAPI. Bar, 5 µm.