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. 2024 Jan 2;43(2):250–276. doi: 10.1038/s44318-023-00018-9

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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/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A–E) HEK293T cells were transfected with the indicated constructs and immunoprecipitated for endogenous UBA6. (A) Empty GFP, and GFP-polyAla with or without a nuclear localization sequence (NLS). (B) WT and mutant PHOX2B (+13Ala). (C) WT and mutant RUNX2 (+6Ala and +12Ala). (D) WT and mutant HOXD13 (+10Ala). (E) WT and mutant PABPN1 (+7 Ala). (F) Cell lysates of HEK293T cells expressing HA-UBA6 and FLAG-USE1 were incubated with recombinant TIG-mutant PHOX2B. Anti-HA beads were used to pulldown UBA6 (beads only were used as control). The bound USE1/UBA6 ratio is shown. n = 3 biological replicates. (G) Cells were transfected with mutant PHOX2B or empty vector. The cells were treated with cycloheximide (CHX) at the indicated times, and were analyzed for E6AP levels. Results are normalized to t = 0. n = 3 biological replicates. (H) Quantification of E6AP mRNA in the cells transfected with empty vector or mutant PHOX2B. n = 5 biological replicates. (I) Control and UBA6-depleted HEK293T cells were transfected with mutant PHOX2B or empty vector and incubated for the last 6 h with the proteasome inhibitor MG132 (10 μM). Endogenous E6AP was immunoprecipitated from cell lysates for ubiquitination analysis. Data information: Data points in (F–H) represent mean ± s.e.m. P values were calculated by paired 2-tailed t test (F), Two-way ANOVA Sidak’s test (G) and unpaired 2-tailed t test (H). *P < 0.05, **P < 0.01, ***P <0.001, ns non-significant. Source data are available online for this figure.