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. 2017 May 10;9:8. doi: 10.3389/fnsyn.2017.00008

Figure 1.

Figure 1

SUMOylation of ectopically expressed activity-regulatedcytoskeleton-associated protein (Arc) and in vitro SUMOylation assay. (A) Ectopically expressed Arc is SUMOylated and forms non-covalent complexes with small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 moieties in HT-1080 fibrosarcoma cell lines. Extract of cells coexpressing Arc with His6-tagged SUMO 1, 2 or 3 were subjected to nickel affinity chromatography and immunoblotted for Arc. A high molecular mass band at ~65 kDa (arrow) corresponding to the covalent addition of single His6-tagged SUMO1, SUMO2, or SUMO3 to Arc was detected. The molecular mass of His6-SUMOylated Arc is higher than physiologically SUMOylated Arc, due to histidine tag residues. The presence of non-modified Arc protein indicates non-covalent complex formation of Arc with His-SUMO modified proteins or free SUMO. (B) Arc was immunoprecipitated from HT-1080 cells expressing non-tagged Arc protein and in vitro SUMOylation was performed with SUMO activating (E1) and conjugating (E2) enzymes in the presence or absence of SUMO1. Upon addition of SUMO1, an Arc immunoreactive band at 65 kDa was detected, corresponding to single SUMO1ylation of Arc. Equal amounts of protein were loaded. No bands were observed in the gel above the 100 kDa marker.