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. 2020 Jan 9;10:83. doi: 10.1038/s41598-019-57053-y

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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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S-SCAM Knockdown reduces the number of GABAergic synapses. Cultured rat hippocampal neurons (div 14) were transfected with plasmids expressing either control shRNA or S-SCAM-specific shRNA (RNAi). After 3 days post-transfection, neurons were fixed and stained for indicated GABA synapse markers. (a) Representative immunofluorescent images of GABA_AR γ2 and vGAT in the dendrites of hippocampal neurons transfected with control or S-SCAM shRNAs. (b) Quantification of puncta densities of GABA_AR γ2 (left), vGAT (middle), and co-localized GABA_AR γ2 and vGAT (right). n = 24 (control) and 23 (S-SCAM RNAi) from triplicate experiments. Unpaired t test. F values are: for GABA_AR γ2, F_(1,45) = 2.128, p = 0.0813; for vGAT, F_(1,44) = 1.543, p = 0.3161; for γ2/vGAT, F_(1,34.9) = 2.653, p = 0.0307. (c) Representative immunofluorescent images of NL2 and GAD65 in the dendrites of hippocampal neurons transfected with control or S-SCAM RNAi. (d) Quantification of puncta densities of NL2 (left), GAD65 (middle), and co-localized NL2 and GAD65 (right). n = 35 (control) and 33 (S-SCAM RNAi). NL2, F_(1,60.31) = 2.140, p = 0.0331; GAD65, F_(1,66) = 1.932, p = 0.0643; NL2/GAD65, F_(1,52.48) = 3.527, p = 0.0005. (e) Representative immunofluorescent images of gephyrin and vGAT. (f) Quantification of puncta densities of gephyrin (left) and co-localized gephyrin and vGAT (right). n = 29 (control) and 27 (S-SCAM RNAi). gephyrin, F_(1,47) = 1.615, p = 0.2545; gephyrin/vGAT, F_(1,54) = 1.572, p = 0.2436. Scale bars, 5 μm.