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. 2010 Jul 27;123(16):2823–2833. doi: 10.1242/jcs.065565

Fig. 2.

Fig. 2.

In vivo and in vitro SUMOylation of Aurora B. (A) HeLa-SUMO2 or parental cells were transfected with UBC9 or V5-Aurora-B-expressing vectors as indicated. Cells were harvested after 18 hours in the presence of taxol or 3 hours after release from taxol. His-SUMO2 conjugates were recovered on Ni-NTA beads and analyzed by immunoblotting (IB). The indicated antibodies were used for immunoblot analysis on total protein lysates (input) or SUMO2 conjugates purified with Ni-NTA beads. The position of V5-Aurora-B-SUMO2 conjugates (~60 kDa and higher) is indicated by brackets. An additional band of ~55 kDa (predicted size for SUMO conjugates with endogenous Aurora B) is detected by the mouse monoclonal antibody against Aurora B. The asterisk indicates an unspecific band detected by the rabbit, but not the mouse, anti-Aurora-B antibody. (B) In vitro SUMOylation of Aurora B. Recombinant p53, Aurora B or Aurora B in complex with the IN-box of INCENP were incubated in the presence of SUMOylation machinery and SUMO2 or a SUMO2 non-conjugatable mutant. Proteins were immunodetected using specific antibodies against the indicated proteins. p53- or Aurora-B-SUMO2 conjugates are indicated by arrows.