(A) HeLa cells expressing GFP alone (3XGFP-NLS) or GFP-Aurora B were arrested in mitosis using Taxol, immunoprecipitated using GFP-Trap beads (GFP-IP) and analyzed by Western blotting. (B) HeLa cells expressing GFP-Aurora B were treated with control (-) or CUL3 siRNAs (+), synchronized and analyzed as in (A). (C) MDA-MB-231 cells were arrested in mitosis by STLC, immunoprecipitated using UBASH3B antibody or IgG and analyzed by Western blotting. The modified and unmodified Aurora B forms are shown (SE: short exposure, LE: long exposure). The asterisk (*) indicates a non-specific signal and IgG HC points to the heavy chain of IgG. (D) Recombinant GST or GST-UBA domain of UBASH3B were incubated with extracts of mitotically synchronized HeLa cells, immunoprecipitated using glutathione-sepharose beads (GST-IP) and analyzed by Western blotting. Arrows indicate the unmodified and ubiquitin-modified Aurora B. (E) Recombinant wild-type (GST-UBA-WT), mutated form with M47A (GST-UBA-Mut), and with W72A/H76A/D79A within extended UBA (GST-eUBA-Mut) were incubated with extracts of mitotically synchronized HeLa cells expressing GFP-Aurora B and analysed as in (D) and by Coomassie blue staining and Western blotting. Arrows indicate unmodified and ubiquitin-modified GFP-Aurora B.