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. 2019 Jul 5;10:2998. doi: 10.1038/s41467-019-10992-6

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© The Author(s) 2019

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. 4 — Inc and Cul3 levels are rapidly enhanced at postsynaptic densities following glutamate receptor perturbation. a Representative NMJ image of endogenously tagged Inc^smFP before and after PhTx application. NMJs are immunostained with anti-GFP (Inc^smFP), the presynaptic membrane marker HRP, and the postsynaptic density marker DLG. Note the increase in Inc^smFP levels at NMJs (labeled by HRP and DLG). b Schematic illustrating the HRP + DLG signal (where Inc^smFP overlaps in both pre- and postsynaptic areas) and the DLG only component (where only postsynaptic Inc^smFP overlaps with DLG). c Quantification of Inc^smFP intensity at specified areas following PhTx application relative to baseline (–PhTx) (–PhTx: inc^smFP, n = 21; + PhTx: inc^smFP, n = 15). d NMJ image of endogenously tagged Inc^smFP with inc expression knocked down in the presynaptic motor neuron using Inc-RNAi (inc^smFP/Y;OK6-Gal4/UAS-inc RNAi) before and after PhTx application. Note that while the Inc^smFP signal that overlaps with HRP is reduced by 52% (Supplementary Table 2), a significant increase in the total level of Inc^smFP at the NMJ is observed. e Quantification of Inc^smFP intensity at NMJs in presynaptic inc knock down following PhTx application relative to baseline (–PhTx) (–PhTx: inc^smFP + MN > Inc RNAi, n = 10; + PhTx: inc^smFP + MN > Inc RNAi, n = 11). f NMJ image of a Flag-tagged Cul3 transgene expressed in the postsynaptic muscle (G14-Gal4/UAS-3xFlag-3xHA-Cul3) before and after PhTx application. Note that Cul3 signals are enhanced at NMJs following 10 min PhTx application. g Quantification of Flag (Cul3) intensity at PSDs (labeled by DLG) following PhTx application relative to baseline (–PhTx) (–PhTx: muscle > flag-Cul3, n = 19; + PhTx: muscle > flag-Cul3, n = 18). Asterisks indicate statistical significance using a Student’s t-test: (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001, (ns) not significant. Error bars indicate ± SEM. n values indicate biologically independent cells. Additional statistical information and absolute values for normalized data can be found in Supplementary Table 2. Source data are provided as a Source Data file