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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. 3 — a THP-1 cells were treated with interferon (IFN)-β for 8 h or left untreated. Lysates were separated by density gradient ultracentrifugation on glycerol gradients (10–40% (v/v), Svedberg constants of the standard markers are indicated above the blots) to isolate the monomeric versus oligomeric form of RNF213. Twenty fractions (frac#) for each sample were collected, concentrated by TCA precipitation and analyzed by immunoblotting against RNF213, showing the presence of oligomer RNF213 in fraction 14–20 upon interferon treatment (upper panel). Alternatively, RNF213 was immunoprecipitated (IP) from each fraction, first desalted over Amicon columns. Immunoprecipitated material was eluted into loading buffer and analyzed by immunoblotting against ISG15 and ubiquitin, showing (hyper)ISGylation of oligomer RNF213 upon interferon treatment (lower panel). b The monomeric and oligomeric forms of RNF213 were separated by density gradient ultracentrifugation after interferon-β treatment as in a and knockdown of UBE1L by siRNA (siUBE1L) treatment for 48 h, using a nontargeting scrambled siRNA (siScramble) as control. Knockdown of UBE1L strongly reduced RNF213 oligomerization upon interferon treatment. c LDs were isolated by ultracentrifugation floatation assay on a sucrose step-gradient and associated proteins were further separated by density gradient ultracentrifugation to isolate the monomeric and oligomeric form of RNF213 after interferon-β treatment as in a. Fractions were concentrated by TCA precipitation and analyzed by immunoblotting against RNF213, showing association of oligomeric RNF213 with LDs upon interferon treatment. Source data are provided as a Source Data file.