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. 2020 Feb 10;10:2268. doi: 10.1038/s41598-020-59005-3

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© The Author(s) 2020

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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SIAH2-mediated downregulation of its interaction partner HO-1. (A) 293T cells were transfected to express increasing amounts of HA-SIAH2 along with Flag-tagged HO-1 as shown. Two days later cells were harvested and analysed by Western blotting as shown. (B) The experiment was done as in (A) with the exception that increasing amounts of HA-SIAH1 were expressed. (C) 293T cells were transfected to express HA-tagged HO-1, either alone or together with wild-type SIAH2 or a SIAH2 RING domain mutant (RM). The relative expression of proteins is shown, please note that the SIAH2 RM shows an increased expression level due to the lack of auto-ubiquitination and degradation. (D) Lysates from 293T cells were used for immunoprecipitation (IP) using antibodies for SIAH2 (upper) or HO-1 (lower) in combination with control IgGs. The precipitated samples were analysed by Western Blotting using appropriate antibodies, input samples ensured correct protein expression.