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. 2020 Jan 29;11:570. doi: 10.1038/s41467-020-14345-6

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© The Author(s) 2020

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. 8 — a Representative FRAP images at different time points of DIV21–23 cortical neurons transfected with EGFP–GluN1 (WT, I272A, or N273A/R337D) together with GluN2B, CRISPR construct targeting endogenous GluN1, and mCherry. Recovery of bleached spine puncta (magenta circle) was monitored for 15 min at 10s intervals. Scale bar = 2 µm. b Quantification of the recovery curve of different GluN1 mutants in DIV21–23 cortical neurons. Graphs represent mean intensity and show fit (****p < 0.0001, Kolmogorov–Smirnov (KS) nonparametric test). Inset: Quantification of the mobile fraction of EGFP–GluN1 spine puncta at 15 min after photobleaching in EGFP–GluN1 mutant transfected cells compared to WT EGFP–GluN1 in DIV21–23 cortical neurons (*p < 0.05, ANOVA; green dots represent WT n = 33 puncta; I272A n = 12; N273A/R337D n = 12). Error bars show S.E.M. c Representative FRAP images at different time points of DIV21–23 cortical neurons transfected with EGFP–GluN1 (WT, WT+EphB2 knockdown, N273A/R337D + EphB2 knockdown, WT+EphB2 knockdown rescued by RNAi insensitive EphB2) together with GluN2B, CRISPR construct targeting endogenous GluN1, and mCherry. FRAP was conducted on serveral puncta in different dendritic branches. Recovery of bleached spine puncta (magenta circle) was monitored for 15 min at 10s intervals. Scale bar = 2 µm. d Quantification of the recovery curve of different GluN1 mutants in DIV21–23 cortical neurons. Graphs represent mean intensity and show fit (****p < 0.0001, Kolmogorov–Smirnov (KS) nonparametric test). Inset: Quantification of the mobile fraction of EGFP–GluN1 spine puncta at 15 min after bleaching in EGFP–GluN1 mutant transfected cells compared to WT EGFP–GluN1 in DIV21–23 cortical neurons (*p < 0.05, ANOVA; green dots represent WT n = 33 puncta; WT+EphB2 K.D. n = 12; N273A/R337D+EphB2 K.D. n = 21; rescue n = 22). Error bars show S.E.M.