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. 2020 Feb 10;10:2259. doi: 10.1038/s41598-020-58264-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Posttranslational modification of recombinant human GLS2 protein with acetylhypusine. (A) Representative spectrum of a GLS2 peptide with acetylhypusine modification. The peptide KEKKCFPKGVDMMAAL (from amino acid 329 to 344 of the GLS2 sequence) appears as a charge + 3 peptide with a monoisotopic m/z of 661.34753 Da (+0.29 mmu/+0.44 ppm). MH: 1982.02805 Da, including C-5 carbamidomethyl (57.02 Da), M-12 oxidation (15.99 Da) and K4-Acetylhypusine (113.08 Da). (B) Validation of GLS2 hypusination by immunoblot analysis using specific rabbit polyclonal anti-hypusine antibodies. Four different fractions containing affinity-purified recombinant human GLS2 from two different GIP-affinity purifications were analyzed. The anti-hypusine antibodies recognized a band in all four lanes corresponding to the known molecular mass of mature GLS2 protein (29). Electrophoresis was done in 12% polyacrylamide Bis-Tris gel with PageRuler Prestained Protein Ladders (Thermo Scientific) (positions of markers indicated at the right end). (C) Positive and negative controls for hypusination. A whole protein extract from mouse brain and rat prefrontal cortex (20 µg each) were employed as positive controls by revealing a band of the hypusinated eIF5A protein at approx. 17 kDa (lanes 1 and 2). Purified human recombinant GLS2_56–602 protein expressed in bacteria was chosen as a negative control. It was analyzed in 10% SDS-PAGE gel and revealed with anti-hypusine antibodies (lane 3) or anti-GLS2 antibodies (lane 4). Positions of molecular mass markers are shown at the left end of panel C. Full-length gels and blots are shown in both panels B and C.