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. 2022 Feb 3;14(2):314. doi: 10.3390/v14020314

Figure 3.

Figure 3

The M1 protein of IBV was SUMOylated and the SUMOylation of the M1 protein was inhibited with the SUMOylation inhibitor STE025 in both vitro FRET assay and the biochemical assay. (A) The FRET spectrum of the in vitro SUMOylation reaction of IBV M1 protein using the FRET assay. Four reactions were conducted, CyPet-SUMO1, E1, E2, E3, YPet-M1, and ATP (ALL and green); CyPet-SUMO1, E1, E2, YPet-M1, and ATP (no PIAS1 and Blue); E1, E2, E3, YPet-M1 and no ATP (NO ATP and black); CyPet-SUMO1, E1, E2, E3, YPet-M1, ATP, and STE (ALL plus STE and red). (B) Quantitative FRET signal (EmFRET) of IBV M1 SUMOylation from (A). (C) In vitro biochemical assay of the SUMOylation of IBV M1 protein followed with a Western blot using anti-SUMO1 antibody. The SUMOylation reactions were conducted in solution in various conditions with or without the SUMOylation inhibitor, STE. Lane 1, CyPet-SUMO1, E1, E2, E3, YPet-M1, -ATP; Lane 2, CyPet-SUMO1, E1, E2, YPet-M1, +ATP; Lane 3, CyPet-SUMO1 1, E1, E2, E3, YPet-M1, +ATP; Lane 4, CyPet-SUMO1, E1, E2, YPet-M1, +ATP+STE025.