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. 2017 Jan 30;7:41734. doi: 10.1038/srep41734

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Copyright © 2017, The Author(s)

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(A,B) Western blotting of the homogenate (HOMO), postsynaptic triton insoluble fraction (TIF) and triton soluble fraction (TSF) obtained from control and FTY720-treated hippocampal neurons (DIV14). Tubulin is used as loading control, while PSD-95 as a marker of postsynaptic fraction. FTY720 leads to an increased GLUN2B localization in the TIF leaving the total amount of GLUN2B unaltered while doesn’t alter GLUN2A localization in TIF (Student’s t-test, P = 0.0275 N = 4). (C) Confocal images of neurons live stained for GLUN2A, fixed and counterstained against β3-tubulin. Relative quantification of density of GLUN2A puncta are shown on the left (Mann-Whitney Rank Sum Test, P = 0.070) and mean intensity of GLUN2A clusters on the right (Mann-Whitney Rank Sum Test, P = 0.001). (D) Images of neurons live stained for GLUN2B and quantification of GLUN2B puncta density (Mann-Whitney Rank Sum Test, P = 0.003) and mean intensity (Student’s t-test, P = 0.610). (E,F) Images of neurons live stained for GLUN2A (E) or GLUN2B (F), fixed and counterstained against the presynaptic marker bassoon. Right histograms show quantification of GLUN2A positive synapses (Mann-Whitney Rank Sum Test, P ≤ 0.001) and GLUN2A puncta mean intensity (Mann-Whitney Rank Sum Test, P = 0.005) (number of analyzed puncta: control = 1010, FTY720 = 720; number of analyzed fields: control = 35; FTY720 = 30) or GLUN2B positive synapses (Mann-Whitney Rank Sum Test, P = 0.005) and GLUN2B puncta mean intensity (number of analyzed puncta: control = 1250, FTY720 = 807; number of analyzed fields: control = 37; FTY720 = 32). (G) Representative images of spines co-transfected with SEP-GluN2A(green) and dTom (red) acquired at the indicated time point in control and FTY720-treated neurons. (H) XY graph representing the DMFI/MFI₀ of SEP-GluN2A over time (number of analysed spines: control = 40, FTY720 = 47; Mann-Whitney Rank Sum Test, P = 0.037 at t₅₀ and P = 0.009 at t₆₀). (I) Images of spines co-transfected with SEP-GluN2B and dTom as in G. (J) XY graph representing the DMFI/MFI₀ of SEP-GluN2B over time (number of analysed spines: control = 15, FTY720 = 22; Mann-Whitney Rank Sum Test, P = 0.030 at t₆₀).