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. 2016 Oct 13;7(10):e2411. doi: 10.1038/cddis.2016.312

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Copyright © 2016 The Author(s)

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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Pro-C3 is ubiquitylated in the livers of refed mice. (a and b) Western blotting after anti-FLAG immunoprecipitation (FLAG IP) (a) and histidin-biotin pulldowns (His-Bio pulldown) (b) of whole-cell lysates from HEK293T cells ectopically expressing FLAG-tagged C3 and His-Bio-tagged ubiquitin. Cells expressing FLAG alone were used as a control. Cells were treated with DMSO, 10 μM of proteasome inhibitor MG132 or 50 μg/ml BFA for 4 h as indicated. Input, FLAG IP or HIS-Bio pulldowns are shown separately. Signals with an antibody against FLAG-tag or revealed by streptavidin conjugated to horseradish peroxidase (Strep-HRP) are shown separately. (c–e) Western blotting after FLAG-IP (c and e) and a His-Bio pulldown (d) of whole-cell lysates from primary murine hepatocytes ectopically expressing FLAG-tagged C3 and His-Bio-tagged ubiquitin. Cells expressing FLAG alone were used as a control. Cells were treated with DMSO, MG132 (c and d) or BFA (e) as indicated. Input, FLAG IP or HIS-Bio pulldowns are shown separately. Signals with an antibody against FLAG tag or revealed by streptavidin conjugated to horseradish peroxidase (Strep-HRP) are shown separately. (f) Western blotting after TUBEs 1 pulldowns of whole-liver lysates from fasted and refed mice as indicated. Input and pulldowns are shown separately. Liver lysates from C3 knockout mice (C3KO) were used as a control. Antibodies against ubiquitin (P4D1) and C3a were used. Arrows indicate endogenous pro-C3 and the alpha chain (α chain) of mature C3