Xenbase Image ID: 125485

Figure 1. PIASy but not RanBP2 is required for SUMO2/3 conjugation of TopoIIα in XEE. (a) XEE were immunodepleted using antibodies against RanBP2, PIASy, or RanBP2/PIASy. The depleted extracts were incubated with 10,000 sperm nuclei/µl for 1 h at 25°C. Non- or mock (IgG)-depleted extracts were also subjected to the same procedure. Isolated chromosomes from each reaction were analyzed by Western blotting (WB) for the indicated protein. Immunodepletion (ID) of RanBP2 had no effect on the SUMOylation of TopoIIα, whereas PIASy ID eliminated TopoIIα SUMOylation. The arrow and bracket indicate unmodified and SUMO2/3-modified TopoIIα, respectively. Positions of molecular mass standards (kD) are indicated on the left. (b) The mitotic chromosomes were prepared as in Materials and methods and were analyzed by immunostaining with the indicated antibodies: TopoIIα is shown in red, PIASy is shown in green, and SUMO2/3 is shown in blue in merged panel. TopoIIα colocalized with PIASy and SUMO2/3 at the centromeres. The addition of dnUbc9 eliminated SUMO2/3 modification but did not alter the localization of TopoIIα at the centromeres of mitotic chromosomes. Bars, 10 µm. Image published in: Ryu H et al. (2010) © 2010 Ryu et al. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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