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. 2021 Jul 13;12:4285. doi: 10.1038/s41467-021-24505-x

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© The Author(s) 2021

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. 7 — a Major domains in neurofibromin (GRD and CRAL-TRIO), site of the R1320P mutation (red arrow), and GFP C-terminal fusion. b Transgenic expression of full-length Nf1 and Nf1^R1320P in the heteroallelic Nf1^P1/E1 mutant background. Full length Nf1 rescues CO₂ production to nSyb-Gal4/+ levels. Experimental (UAS-Nf1; nSyb-Gal4, Nf1^P1/E1) ***p = 0.001 re: Nf1^P1/E1, **p = 0.009 re: UAS-Nf1; Nf1^P1/E1, **p = 0.005 re: nSyb-Gal4, Nf1^P1/E1, ***p = 0.0003 re: UAS-Nf1^R1320P; nSyb-Gal4, Nf1^P1/E1. (Dunn’s test, two-sided; n = 8 nSyb-Gal4/+, n = 8 Nf1^P1/E1, n = 6 UAS-Nf1; Nf1^P1/E1, n = 7 nSyb-Gal4, Nf1^P1/E1, n = 8 UAS-Nf1; nSyb-Gal4, Nf1^P1/E1, n = 7 UAS-Nf1^R1320P; nSyb-Gal4, Nf1^P1/E1). c Representative western blot of anti-GFP and β-tubulin shown. Samples derived from the same experiment and processed in parallel. Blot normalized to the intensity of loading control (β-tubulin). Quantification represents four replicates. au: arbitrary units, ns: not significant. p = 0.510 (Student’s t-test, two-sided; n = 4 per genotype). d Representative western blot of Nf1 and pERK levels in Nf1^P1 and Nf1 RNAi flies compared to genetic controls (w^CS10 and Gal4/+, respectively). Samples derived from the same experiment and processed in parallel, with β-tubulin as loading control on the same blots. Representative blot shown from one of two independent experiments. e Normalized CO₂ production with expression of an ERK gain-of-function mutation (UAS-ERK^Sem) in PCB-Gal4. ***p < 0.001 (Sidak, two-sided; n = 10 per genotype). Each “n” = in (b, e) represents a single respirometer (containing 4 animals). Box plots—box: 1st to 3rd quartiles, median: line, whiskers: min–max, individual data points: circles.