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. 2018 Jan 23;8:1410. doi: 10.1038/s41598-018-19471-2

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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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Ser-385 phosphorylation modifies HACE1 properties in cells. (a) Cytosol and membrane fractions from MCF12A cells transfected with HA-HACE1 and myc-Rac1(Q61L) were analyzed by immunoblot (IB) using the indicated antibodies. Anti-RhoGDIα and anti-Transferrin-R antibodies are used as specific markers of cytosol and membrane fraction, respectively. (b) Protein lysates from MCF12A cells transfected with the indicated plasmids were subjected to His pull-down (His-PD) prior to immunoblot analysis (IB). Whole cell lysate (WCL) IB analysis showing total protein expression. (c) Protein lysates from MCF12A cells transfected with the indicated plasmids were subjected to immunoprecipitation (IP) using Ctrl or HA antibodies prior to immunoblot analysis (IB). Whole cell lysate (WCL) IB analysis shows total protein expression. (d) In vitro ubiquitination assay using recombinant 6His-tagged HACE1(WT), catalytic inactive mutant C876S (CS) and HACE1(S385E) (SE) analyzed 30 min post-reaction by immunoblot using the indicated antibodies. IB at t = 0 min shows the input protein levels. (e) In vitro ubiquitination assay using HACE1(WT) and HACE1(S385E) (SE) analyzed by IB at the indicated time points. (f) In vitro ubiquitination assay using recombinant 6His-tagged HACE1(WT), catalytic inactive mutant HACE1(C876S) (CS), HACE1(S385A) (SA), HACE1(S385E) (SE) and Rac1 loaded with GTPγS and analyzed 30 min post-reaction by immunoblot using the indicated antibodies. IBs in (a–f) are cropped from the full-length blots shown in Supplemental Figure S12.