Abstract
This chapter deals with the fluorescence detection of SUMOylation and deSUMOylation in semi-intact cultured human cells, the so-called "in situ SUMOylation assay" and the "in situ deSUMOylation assay," respectively. In the in situ SUMOylation assay, the recombinant green-fluorescence protein fused to the SUMO1 (GFP-SUMO1) protein is used to visualize the nuclear rim, nucleolus, and nuclear bodies. These GFP signals represent cellular regions where SUMOylation efficiently takes place. If the recombinant SUMO-specific protease SENP1-catalytic domain is added after in situ SUMOylation, GFP signals can be erased. Therefore, the in situ SUMOylation assay can be used to assess deSUMOylation enzymatic activity.
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