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. 2024 Feb 28;627(8005):873–879. doi: 10.1038/s41586-024-07112-w

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 9 — a, Left panels: bio-layer interferometry binding assays of His-Halo-tagged cGAS mutants with SPSB3-ELOBC. Right panels: binding affinity of cGAS mutants with SPSB3-ELOBC. b, Bio-layer interferometry binding assays His-Halo-tagged cGAS with an SPSB3 mutant in complex with ELOBC. c, Immunoblot showing in vitro ubiquitylation reactions of wild-type human cGAS catalytic domain (CD; aa: 155–522) or distinct cGAS CD mutants by CRL5^SPSB3. d, Cell lysates from HeLa cells, HeLa cGAS KO cells reconstituted with doxycycline-inducible wild-type cGAS (WT), cGAS(NN) treated with different concentrations of doxycycline for 3 days were analysed by immunoblot. Vinculin was used as a loading control. e, IP of GFP-tagged cGAS from HEK293T cells transfected with constructs for GFP-tagged NLS-cGAS (WT) or GFP-tagged cGAS(NN), HA-tagged ubiquitin, and SPSB3. Samples were analysed by immunoblot. f, Relative nuclear cGAS-GFP MFI measurement in post-mitotic HeLa cGAS KO cells reconstituted with cGAS (WT) or cGAS(NN) (n = 15 cells per condition). Data are mean ± SD. Two-way ANOVA with Šídák’s multiple comparison test was used for statistical analysis. One representative of two (d-e) or three (a-c) independent experiments is shown.

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