Figure 2.

Aurora-A co-localizes with UBC9 and SUMO1 at the spindle and is SUMOylated both in vitro and in vivo. (A) Aurora-A and UBC9 mainly localize at interphase centrosomes and onto the mitotic spindle. HeLa cells were fixed and processed for immunodetection of Aurora-A (red) and UBC9 (green) and observed under a microscope at the indicated phases of the cell cycle. The Aurora-A and UBC9 signals were coincident on the centrosomes and the nuclear membrane in interphase, at the spindle in metaphase and on the cytokinesis bridge at the end of telophase. Note that whereas Aurora-A is still present at the spindle during anaphase and telophase (white arrowheads), this is not the case for UBC9. By contrast, UBC9, and not Aurora-A, is clearly visible at the midzone during anaphase (yellow arrowheads). (B) Co-localization of Aurora-A and SUMO1. Endogenous SUMO1 (red) and Aurora-A (green) were detected by immunofluorescence in HeLa cells. Both proteins co-localize at the spindle (poles and microtubules) during metaphase. (C) Recombinant Aurora-A protein was used as substrate in an in vitro SUMOylation assay in the presence of SUMO1 or SUMO1 mut (a non-conjugatable SUMO1 mutant). Proteins were separated by reducing 12% SDS-PAGE and visualized with Coomassie Blue (left panel). Reaction products were used to detect both Aurora-A and SUMO1 in western blot (right panels). Black arrows indicate unSUMOylated Aurora-A, red arrows indicate SUMOylated Aurora-A, and blue arrows mark other proteins included in the reaction that are also modified by SUMO1. (D) HeLa cells were co-transfected with the indicated expression vectors. Forty-eight hours later proteins were isolated and SUMO1 were conjugates purified under denaturing conditions. V5-tagged Aurora-A was detected by immunoblotting in both the inputs and Ni-NTA purified fractions, whereas vinculin was immunodetected only in the inputs. The V5–Aurora-A signal in the Ni–NTA fractions was quantified after normalization with the inputs signals for V5–Aurora-A.