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. 2019 Jan 30;9:951. doi: 10.1038/s41598-018-37264-5

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© The Author(s) 2019

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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USP19 specifically modulates EWS-FLI1 protein levels. (a,b) Immunoblot analysis of USP19 depleted cells. (a,b) A673 and SKNMC cells were transiently transfected with 20 nM siRNAs for 72 h as indicated. Lysates were subjected to western blot analysis and analyzed by anti-FLI1, anti-USP19 and anti-p27 antibodies. Arrows indicate specific USP19 isoforms, asterisk marks an unspecific band. Right panel to each western blot, quantification of EWS-FLI1 proteins levels (n = 5–6, the mean is indicated by the horizontal line, error bars as SD). (c) Active USP19 stabilizes EWS-FLI1 protein. 3xflag-EWS-FLI1 was transiently co-expressed with a control vector or increasing levels (ratios 3xflag-EWS-FLI1 to 3xmyc-USP19 1:2 and 1:4) of wild-type or catalytically inactive 3xmyc-USP19 for 48 h in HEK293T cells. Lysates were analyzed by western blotting using anti-flag and anti-myc antibodies. (d) Quantification of 3xflag-EWS-FLI1 protein levels of (c) with n = 8 for control and n = 4 for others, the mean is indicated by the horizontal line and error bars as SD. (e) USP19 overexpression stabilizes specifically EWS-FLI1. 3xflag-EWS-FLI1, 3xflag-EWSR1 and 3xflag-FLI1 were transiently co-expressed with increasing concentrations (ratios 3xflag-protein to 3xmyc-USP19 1:2 and 1:4) of active 3xmyc-USP19 for 48 h in HEK293T cells. Lysates were analyzed by western blotting using anti-flag and anti-myc antibodies. Numbers below represent densitometrically quantified flag tagged protein over loading control GAPDH of a representative experiment.