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Figure 6


Fig. 6. Exchange dynamics of SP100 and SP100 mutant constructs at NBs. (A) Schematic depiction of SP100 protein and mutants used for FRAP analyses. The oligomerization and PML NB targeting resides within a region spanning amino acids 29-152 of SP100 (Negorev et al., 2001). SP100 has a SUMOylation site at Lys297 (S) within the HP1-binding motif and a nuclear localization signal at the C-terminus (NLS). Note, that SP100 variants that lack the endogenous NLS were engineered to contain one at their N-termini (Negorev et al., 2001). (B) FRAP experiments were performed in U-2 OS cells that express GFP-tagged SP100 wild-type (aa 1-480) and the indicated variants, by bleaching areas that contain NBs and by monitoring fluorescence recovery for various intervals as indicated. Scale bars, 5 µm. (C,D) Quantification of FRAP experiments as shown in B. Graphs show mean values (± s.d.) from at least 20 FRAP experiments. For comparison, the FRAP curve of GFP–PML-IV at NBs was included in C.





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