Fig. 1: Aurora B is modified by K63-linked ubiquitination in mitosis. | Communications Biology

Fig. 1: Aurora B is modified by K63-linked ubiquitination in mitosis.

From: The deubiquitinating enzyme complex BRISC regulates Aurora B activation via lysine-63-linked ubiquitination in mitosis

Fig. 1

a K63-linked ubiquitination (K63-Ub) of ectopic Aurora B in mitotic HEK293T cells. Cells were co-transfected with Flag-HA-Aurora B (FH-Aurora B) and His-K63-Ub, and arrested in mitosis with 30 ng/mL nocodazole treatment for 16 h. Whole-cell lysates (WCL) were subjected to immunoprecipitation (IP) using anti–FLAG M2 agarose beads and IP samples were analyzed by immunoblotting (IB) using an antibody specifically recognizes K63-Ub chains. S.E., short exposure; L.E., long exposure. b K63-Ub modification of endogenous Aurora B in mitotic HeLa cells. HeLa cells were synchronized to mitotic (M) phase as described in (a), then subjected to IP using anti-Aurora B antibody followed by IB. c, d Confirmation of the K63-Ub modification of Aurora B under denaturing condition. HEK293T cells were transfected with V5-His-Aurora B and wild-type ubiquitin or with ubiquitin mutants containing only one Lys (c), Ub-K48R or Ub-K63R (d). Mitotic cells were subjected to Ni-NTA pull-down under denaturing conditions and the immunoprecipitates were analyzed by indicated antibodies.

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