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. 2018 Jul 6;131(13):jcs214924. doi: 10.1242/jcs.214924

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© 2018. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 2. — Verification of Psm1-K536 methylation by anti-Psm1-K536me antibody. (A) Specificity of antibody generated against Psm1-K536me. The indicated amounts of Psm1 peptide CTPTQKK(me)YESAIA carrying methylation on K536 (K536me), an unmodfied peptide CTPTQKKYESAIA (unmodified) or modified peptide CTPTQKAYESAIA (K536A) were spotted on PVDF membrane and probed with anti-Psm1-K536me antibody or pre-immune serum. (B) Psm1–Pk protein was immunoprecipitated (IP) from cycling wild-type cells (Psm1–Pk) or mutant cells carrying the psm1^K536A mutation (Psm1-K536A–Pk). Samples were eluted in two steps and both were analyzed using anti-Pk antibody. Eluate 1 from each sample was analyzed with anti-Psm1-K536me antibody. The amount of loaded samples is indicated. The same protein extracts were analyzed on two different gels. (C) Nuclear spreads were prepared from cycling wild-type cells (Psm1) or mutant cells carrying the psm1^K536A mutation (Psm1-K536A). Shown are selected nuclei after chromosome spreading, staining with DAPI and after immunolabeling with anti-Psm1-K536me antibody.