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. 2017 Oct 3;3:17035. doi: 10.1038/celldisc.2017.35

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Copyright © 2017 The Author(s)

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LSD1 demethylates YY2 K247 methylation in vitro. (a) YY2 (227–372) was pre-methylated by SET7/9, and then in vitro demethylation assay was performed by adding purified bacterially expressed His-tagged LSD1 wild type (wt) or its enzymatically dead mutant (m), followed by autoradiogram (top panel). Gel intensity was quantified by using Image J (National Institute of Health, Bethesda, MD, USA) and was shown as indicated. The expression of LSD1 (wt), LSD1 (m), SET7/9 and YY2 (227–372) was examined with C.B.S. (bottom panel). (b) In vitro demethylation assay was performed as described in a by adding increased amount of LSD1 (wt), followed by autoradiogram (top panel). Gel intensity was quantified by using Image J and was shown as indicated. The expression of LSD1 (wt), SET7/9 and YY2 (227–372) was examined with C.B.S. (bottom panel). (c) In vitro demethylation assay was performed by mixing SET7/9-pre-methylated K247 peptide and LSD1 (wt) or LSD1 (m), followed by dot blot assay and autoradiogram. The expression of LSD1 (wt) and LSD1 (m) was examined with C.B.S. (bottom panel). (d) In vitro demethylation assay was performed by mixing synthetic short peptides from YY2 containing monomethylated K247 (YY2K247me1) with or without purified bacterially expressed LSD1 protein, followed by MALDI-TOF MS analysis. YY2 K247 peptide (unmodified) served as a control.