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. 2018 Oct 22;9:4385. doi: 10.1038/s41467-018-06854-2

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© The Author(s) 2018

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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Fig. 6 — Dual role of UBE2E family in regulating ASXL2 protein stability. a Depletion of UBE2E3 results in increased DEUBAD (ASXL2) protein levels. U-2 OS cells stably expressing GFP-DEUBAD (ASXL2) were transfected with a different siRNAs oligos for UBE2E3 and then treated with CHX as indicated. Right panel, densitometry analysis of DEUBAD protein bands was conducted and presented as indicated. n = 3 biological replicates. b Combined depletion of all three UBE2Es resulted in a stronger increase of DEUBAD (ASXL2) protein levels which become similar to those of GFP-DEUBAD K370R. U-2 OS cells stably expressing GFP-DEUBAD (ASXL2) were transfected with Non-target siRNA control or a combination of UBE2Es (UBE2E1, UBE2E2, UBE2E3) siRNAs, treated with CHX as indicated and used for immunoblotting. Right panel, densitometry analysis of the protein levels of DEUBAD bands were conducted and presented as in a. n = 3 biological replicates. c Opposite effects of UBE2Es on DEUBAD (ASXL2) and ASXL2 protein levels depending on BAP1 expression. Flag-UBE2Es expression constructs were transfected in HEK293T cells in the presence of Myc-DEUBAD (ASXL2) or Myc-ASXL2 expression vectors in presence or not of BAP1 and cells were harvested for immunoblotting. n = 3 biological replicates. d Depletion of UBE2E1, UBE2E3 and the combination of three UBE2Es resulted in decreased protein levels of endogenous ASXL2 and BAP1. U-2 OS cells were transfected with Non-target control (NT) or UBE2E siRNAs and then treated with CHX and used for immunoblotting. Bottom panel, densitometry analysis of ASXL2 and BAP1 levels was conducted and presented for each siRNA. n = 3 biological replicates. e UBE2Es-mediated DEUBAD monoubiquitination is conserved in Drosophila. S2 cells were transfected with dsRNA for Calypso or UBCD2 and Myc-V5-DEUBAD (Asx). DEUBAD monoubiquitination was evaluated by immunoblotting. Bottom panel, densitometry analysis of the protein levels of monoubiquitinated form versus non-modified form of DEUBAD (Asx). n = 3 biological replicates. The stars in panels a, b, and d indicate UBE2E2 band detected with UBE2E1 antibody. Tubulin was used as a loading control for panels a–d and histone H3 was used as a loading control for panel e. Error bars in panels a, b, d, e represents s.d. (mean ± SD)