S-Affs do not inhibit SUMO conjugation or deconjugation.

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S-Affs do not inhibit SUMO conjugation or deconjugation. S-Affs do not inhibit SUMO conjugation or deconjugation. (A and B) In vitro SUMOylation in the presence of the indicated S-Affs for SUMO-1 (A) or SUMO-2 (B) conjugation to GST-tagged RanGAP1 fragment (RG1; amino acids 418 to 587 encompassing a consensus SUMOylation site). Experiments were performed in the presence of increasing concentrations of S-Aff (0.1:1, 1:1, or 10:1 ratios of S-Aff:SUMO). GFP-Adh is a control Affimer raised against GFP and does not bind to SUMO. Blots were probed with an antibody recognizing GFP. (C) SUMOylation of FLAG-tagged PML-I in HEK293T also expressing the indicated FLAG-tagged S-Affs. SUMOylation was induced with As. Proteins were detected with the antibody recognizing the FLAG tag or with an antibody recognizing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (D and E) Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) showing in vitro assays to assess SUMO-1 (D) or SUMO-2 (E) deSUMOylation by the SUMO deconjugases SENP1 and SENP2. All data are representative of at least two independent experiments. David J. Hughes et al., Sci. Signal. 2017;10:eaaj2005 ©2017 by American Association for the Advancement of Science