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. 2018 Feb 14;8:2996. doi: 10.1038/s41598-018-20448-4

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© The Author(s) 2018

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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Synaptic VPS35 is functionally knocked down by three independent shRNA. (a) Representative confocal microscopy images of cortical neurons infected with control shRNA and the three shRNAs against VPS35. Left, mCherry signal reporting the expression of lentivirus containing the shRNAs coding sequences. Right, neurons immunolabeled for VPS35. Scale bar = 50 μm. (b) Representative western blot showing the knock down of VPS35 by three independent shRNAs. Original uncropped blots are shown in Figure S1. (c). Quantification of VPS35 intensity in immunostainings (n = 25 ± 1 fields of view, N = 2 animals) (d) Quantification of VPS35 levels normalized to total protein levels (assessed by TCE staining) in western blot. Values are presented as a ratio compared to the control condition. (N = 5 ± 1 blots/animals). (e) Representative confocal microscopy images of hippocampal neurons expressing either control shRNA or one of the three shRNAs against VPS35. Left, mCherry signal which reports expression of lentivirus containing the shRNAs. Right, surface immunolabelling of GluA1. Scale Bar = 5 μm. (f) Quantitative analyses of GluA1 staining intensity (n = 35 ± 1 fields of view, N = 3 animals). Detailed information (average, SEM, n and statistics) is shown in Supplementary Table S1.