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. 2023 Feb 6;14:638. doi: 10.1038/s41467-023-36298-2

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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. 2 — a Top: Domain organization of p97 and indicated mutations, Middle: Split luciferase assay to measure contacts in HEK293T cells transfected with siRNAs to p97 and indicated N-Myc siRNA-resistant rescue constructs, RLU relative luminescence unit, Bottom: Immunoblot of indicated proteins (n = 9, 7, 8, 5, 3, 3, 3, and 3 biologically independent samples from left to right, respectively). b Top: Domain organization of UBXD8 and indicated mutations, Middle: Split luciferase assay to measure contacts in HEK293T cells transfected with siRNAs to UBXD8 and indicated C-HA/FLAG siRNA-resistant rescue constructs, RLU relative luminescence unit, Bottom: Immunoblot of indicated proteins. UBA ubiquitin associated, UAS upstream activating sequence, UBX ubiquitin X. (n = 10, 6, 10, 7, 4, 6, & 4 biologically independent samples from left to right, respectively). c Top: Split luciferase assay to measure contacts in HEK293T cells transfected with the indicated siRNAs against the p97 adapters. (n = 8, 6, 3, 3, 5, 3, and 8 biologically independent samples from left to right, respectively). Bottom: Immunoblot of HEK293T cells transfected with indicated siRNAs. d Top: Split luciferase assay to measure contacts in HEK293T cells transfected with two independent siRNAs against HRD1 or in HEK293 WT or GP78 KO cells. (n = 3 biologically independent samples of siControl, siHRD1#3, and siHRD1#4; n = 4 biologically independent samples of WT and GP78 KO). Bottom: Immunoblot of HEK293T cells transfected with indicated siRNAs. Data are means ± SEM. *, **, ***P < 0.05, 0.01, 0.0001 respectively, ns: not significant, one-way ANOVA with Tukey’s multiple comparison test (a, b, d: siControl vs siHRD1); one-way ANOVA with Dunnett’s multiple comparisons test (c); Two-tailed paired t test for d: HEK293 WT vs GP78 KO #1B. Source data are provided as a Source data file.