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. 2021 Oct 1;12:5772. doi: 10.1038/s41467-021-26061-w

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© The Author(s) 2021

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. 2 — a FLAG immunoprecipitation (IP) was performed from lysates of HEK293T cells expressing FLAG-RNF213 or FLAG-eGFP in combination with HA-ISG15(AA) and the ISGylation machinery (E1, E2, E3). A smear of ISGylated co-immunoprecipitated proteins was detected with FLAG-RNF213, but not with FLAG-eGFP or when nonconjugatable HA-ISG15AA was used. b. THP-1 or primary human monocytes (CD14+) cells were cultured in the presence of 10 mM BSA-conjugated oleic acid and either treated with 10 ng/mL interferon (IFN) -β for 8 h or left untreated. Lipid droplets (LDs)-enriched fractions were isolated by ultracentrifugation floatation assay on a sucrose step-gradient. Immunoblot (IB) against RNF213 and ISG15 revealed an interferon-induced upregulation of both proteins on LDs and a smear of ISGylated proteins associated with LDs. Immunoblots against PLIN1, PLIN2, ATGL, and GAPDH confirmed LD isolation and equal protein loading in the lysate and LD-enriched fraction (1/20th of the lysate and all of the LD-enriched material was loaded). c Similarly, LDs were isolated from THP-1 cells after knockdown of RNF213 by siRNA (siRNF213) treatment for 48 h or using a nontargeting scrambled siRNA (siScramble) as control. Immunoblotting against ISG15 revealed a smear of ISGylated proteins associated with LDs only when RNF213 was present. Immunoblotting against RNF213 confirmed knockdown of RNF213, while PLIN1, PLIN2, ATGL, and GAPDH validated LD isolation and equal protein loading in the lysate and the LD-enriched fraction (1/20th of the lysate and all of the LD-enriched material was loaded). Source data are provided as a Source Data file.