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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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Figure EV4 — (A–C) Mouse primary cortical neurons were transduced with lentiviral vectors expressing GFP-tagged WT PHOX2B or mutant PHOX2B (+7Ala or +13Ala). (A) Quantification of the association of GFP-PHOX2B with the nucleus (Pearson’s coefficient) is presented as well as GFP-PHOX2B cytoplasmic intensity, related to Fig. 4C. (B) Analysis of E6AP and Arc levels in the WT and mutant PHOX2B-expressing neurons (n = 5 and n = 4 biological replicates, respectively). (C) Quantification of Arc intensity in the GFP-PHOX2B expressing neurons is presented (image scale bar 10 μm) as well as a blot showing Arc protein levels. Results represent the average values from neurons in different imaged fields. n = 20, n = 30 and n = 100 neurons analyzed for WT PHOX2B, mutant PHOX2B +7Ala and +13Ala, respectively. (D–F) The neurons were transduced with lentiviral vectors expressing GFP-WT PHOX2B, GFP-mutant PHOX2B (+13Ala) with or without lentiviruses encoding for mCherry-UBA6. (D), E6AP levels were analyzed in cell lysates. Results are normalized to WT PHOX2B. n = 4 biological replicates. (E, F) Quantification of Arc intensity in cycloheximide-treated GFP-PHOX2B expressing neurons that were positive or negative to mCherry. n = 110, n = 150 and n = 50 neuronal cell bodies analyzed for WT PHOX2B, mutant PHOX2B and mutant PHOX2B + UBA6, respectively. Inset shows mCherry signal. Scale bar 10 μm. AU arbitrary units. Data information: Data points in (A–D, F) represent mean ± s.e.m. P values were calculated by one-way ANOVA Tukey’s test (A–D, F). *P < 0.05, **P < 0.01, ****P < 0.0001, ns non-significant.