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. 2021 Nov 18;12:6715. doi: 10.1038/s41467-021-27025-w

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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. 3 — a–d Representative confocal images of fixed dissociated hippocampal neurons co-expressing eGFP, Tet3G/tRNA^Pyl and a pTRE3G-BI PylRS/γ2 (upper panel), pTRE3G-BI PylRS/γ2 S44* (lower panel), d pTRE3G-BI PylRS/γ8 (upper panel), or pTRE3G-BI PylRS/γ8 S72* (lower panel) in the presence of 250 µM TCO*A and 100 ng mL⁻¹ doxycycline live stained with 0.5 µM Pyr-Tet-ATTO643. b, d 20 µm and b, e 4 μm (magnified images). c, f Line scan measurements of Pyr-Tet-ATTO643 across spines and dendritic shaft based on eGFP signal represented in b and e. g Average spine to extraspine intensity ratio of the ncAA staining indicating a spine enrichment of 2.60 ± 0.69 folds for γ2 S44* (blue), and of only 1.17 ± 0.25 fold for γ8 S72* (red). Statistical significance was analyzed using a two-tailed unpaired Welch’s t test; *p < 0.05. h Plot of all the analyzed spines fluorescent intensities as a function of the intensity in a corresponding neighboring equivalent extraspine area in the dendrite for γ2 S44* (blue) and γ8 S72* (red) expressing neurons. The Pearson’s correlation coefficients are γ2 S44*: blue, r = 0.57, p < 0.0001, 872 spines; and, γ8 S72*: red, r = 0.81, p < 0.0001, 521 spines. i Plot of the average ratio per neuron of spine to extraspine intensities as a function of the sum of spine and extraspine intensities for γ2 S44* (blue, r = 0.13, p = 0.521) and γ8 S72*: and γ8 S72* (red, r = 0.−26, p = 0.189) expressing neurons. (g–i) Data relative to γ2 S44* and γ8 S72* pulled from 28 cells each from three independent biological replicates. All representative images are representative of three independent preparations. Source data are provided as a Source Data file.