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. 2022 Sep 21;12(9):220179. doi: 10.1098/rsob.220179

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© 2022 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 2. — AASS activation by N-ethylmaleimide. (a) Effect of different concentrations of DTT (n = 3 for each concentration), THP (n = 2) and NEM (n = 4) on activity of the isolated LOR enzyme. (b) Time dependency of LOR activation by NEM. LOR was preincubated with the indicated concentration of NEM for 0, 15 or 30 min after which the reaction was started with 2-oxoglutarate. Each condition was tested in duplicate. (c) AASS-Myc-DDK activation by NEM. AASS-Myc-DDK was preincubated with the indicated concentration of NEM for 0 or 30 min after which the reaction was started with 2-oxoglutarate. Each condition was tested in triplicate. (d) Immunoblots of purified LOR treated for 10 minutes with the indicated concentration of NEM using anti-N-ethylmaleimide cysteine (OX-133) and anti-AASS antibodies. (e) Effect of different concentrations of NEM (n = 4) on activity of the isolated WT, p.C414S and p.C414Q LOR enzymes. (f) Immunoblots of purified WT, p.C414S and p.C414Q LOR enzymes treated for 10 minutes with 4 µM of NEM using anti-N-ethylmaleimide cysteine (OX-133) and anti-His tag antibodies.