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. 2023 Jan 10;14:79. doi: 10.1038/s41467-022-35608-4

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© The Author(s) 2023

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. 1 — a Schematic drawing of the SDD1 model mRNA used in the in vitro translation (IVT) assay (Top). Schematic of the in vitro experiments (bottom). b, c The purified RNCs in the IVT reaction using HEL2-containing or hel2-knockout (hel2∆) IVT extract were separated by sucrose density gradient centrifugation and detected by UV absorbance at a wavelength of 260 nm. HA-tagged uS10 in each fraction was detected by immunoblotting using an anti-HA antibody. Immunoblotting of purified RNCs using anti-HA (d), K63-linkage (e), and K48-linkage-specific anti-ubiquitin antibodies (f). g In vitro ubiquitination assay. Purified RNCs derived from the hel2-knockout (hel2∆) IVT reaction mixed with Hel2 (E3), Uba1 (E1), Ubc4 (E2), ATP, and ubiquitin or the indicated ubiquitin mutants. After the reaction, HA-tagged uS10 was detected by immunoblotting using an anti-HA antibody. All experiments were performed at least twice with highly reproducible results.