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. 2019 Jan 17;8:e42426. doi: 10.7554/eLife.42426

Figure 3. TRIM28 SUMOylates many transcription factors and transferases.

(A) Schematic of global site-specific SUMO-MS. His-tagged SUMO mutants were co-overexpressed with UBC9 and TRIM28. The SUMOylated proteins were enriched by His-tag beads and separated by SDS-PAGE. Gel fragments were excised and subjected to separate in-gel digestions. The digested peptides were desalted and analyzed by nanoscale LC-MS/MS. (B) SUMOylated proteins were analyzed with STRING. The network were further analyzed by MCODE. Twelve highly interconnected functional subclusters were extracted and shown in different colors. (C) Transferases and transcription factors were clustered by k-means clustering and visualized with STRING analysis. (D) Ten HA-tagged various transcriptional factors were overexpressed with Flag-tagged SUMO proteins, UBC9 and TRIM28. The targeted proteins were immunoprecipitated (IP) by anti-HA-tag beads followed by immunoblotting (IB) with anti-HA and –Flag antibodies. Asterisk (*) indicated the SUMOylated bands.

Figure 3.

Figure 3—figure supplement 1. STRING, MCODE and GO analysis of proteins SUMOylated by TRIM28.

Figure 3—figure supplement 1.

(A) Twelve subclusters, which were extracted by MCODE analysis, were separately plotted. The STRING and MCODE analyses were performed with the following settings: a significance threshold below 10−7, interaction confidence of 0.7, a degree cutoff of 3, a node score cutoff of 0.1, a maximum depth of 2, a K-Core of 5, and haircut. Cluster name and corresponded interconnectivity score were shown below each cluster. (B) Biological process analysis, molecular function analysis, cellular component analysis and protein class analysis were conducted for the identified SUMOylated proteins.