NuMA is a substrate for BRISC DUB activity. (A) Ubiquitination of NuMA increased upon inhibition of BRISC. 293T cells cotransfected with His-tagged NuMA and HA-K63Ub were simultaneously treated with control, ABRO1, or BRCC36 siRNAs. Cell lysates were subjected to Ni-NTA pull-down assays under denaturing conditions. His-NuMA pull-downs were then analyzed by immunoblotting with the indicated antibodies. (B) Endogenous K63-linked ubiquitination of NuMA was increased in ABRO1-silenced HeLa cells. HeLa cells were transfected with control or ABRO1 siRNAs and synchronized at G2/M phase. Cell lysates were subjected to immunoprecipitation by using anti-NuMA antibody and probed with the indicated antibodies. (C) Immunoprecipitations from mitotic HeLa cells by using anti-NuMA antibody were separated by SDS-PAGE and silver stained. (D) Purified GST, GST–ABRO1/BRCC36 WT or GST–ABRO1/BRCC36 QSQ proteins from Sf9 insect cells were separated by SDS-PAGE and stained using Coomassie brilliant blue. Lane 1, GST; lane 2, GST–ABRO1/BRCC36 WT; lane 3, GST–ABRO1/BRCC36 QSQ. (E) In vitro DUB assay. Endogenous NuMA immunoprecipitations, as described in C, were incubated with the purified GST–BRCC36 WT or GST–BRCC36 QSQ complex, as described in D, in DUB buffer. The abundances of K63Ub decreased significantly with the incubation in the presence of GST–BRCC36 WT, but not the inactive GST–BRCC36 QSQ complex.