A) HeLa cells were infected with a lentivirus encoding FLAG-SUMO-2. Cells stably expressing low levels of FLAG-SUMO-2 were selected by flow cytometry. Total lysates were analyzed by immunoblotting to confirm the expression of FLAG-SUMO-2. Ponceau-S staining is shown as a loading control.
B) Stable cell lines were investigated by Z-stacked confocal fluorescent microscopy to confirm nuclear localization of FLAG-SUMO-2. Characteristic SUMO nuclear bodies are indicated with arrows. Upon MMS treatment (0.02% for 90 minutes), SUMO nuclear bodies dispersed. Scale bars represent 5 μm.
C) Schematic representation of the SILAC proteomics workflow. One set of parental HeLa cells and two sets of HeLa cells expressing FLAG-SUMO-2 were differentially SILAC labeled (K0R0/K4R6/K8R10). One labeled FLAG-SUMO-2 set was treated with 0.02% MMS for 90 minutes. The experiment was repeated with reversal of SILAC labels.
D) Coomassie analysis of total lysate sample versus FLAG-purified SUMOylated proteins, prior to in-gel digestion and analysis by mass spectrometry.
E) Schematic representation of the label-free proteomics workflow for identification of SUMO sites. Four sets of His10-SUMO-2-K0-Q87R cells were treated with 0.02% MMS for 90 minutes, and four sets were control treated, prior to SUMO site enrichment, identification and quantification.