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. 2019 Oct 28;17(10):e3000466. doi: 10.1371/journal.pbio.3000466

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© 2019 Ribeiro et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 4 — (A) DIV8 to DIV10 WT mouse cortical neurons transfected with WT HA-Nrxn1α and a cytoplasmic deletion mutant lacking the 4.1-binding motif (HA-Nrxn1α Δ4.1) and immunostained for surface (s.) HA-Nrxn1α (grayscale). Red arrowheads indicate the axon, and the blue asterisk marks the cell body. High-zoom images show dendritic (D, dotted blue box) and axonal (A, dotted red box) HA-Nrxn1α. (B) Quantification of panel A: surface HA-Nrxn1α fluorescence intensity in axon and dendrites relative to total surface levels and normalized to cells expressing WT-Nrxn1α and ratio of axonal–dendritic surface HA intensity. WT (n = 30 neurons); Δ4.1 (n = 29). ***P < 0.001 (Mann-Whitney test, 3 independent experiments). (C) DIV8 to DIV10 mouse Sorcs1^flox/flox cortical neurons electroporated with EGFP (Ctr) or Cre-EGFP (Cre) and transfected with WT HA-Nrxn1α or HA-Nrxn1α Δ4.1. Neurons were immunostained for surface HA-Nrxn1α (grayscale). (D) Quantification of panel C: ratio of axonal–dendritic surface HA-Nrxn1α fluorescence intensity. Ctr_WT (n = 46 neurons); Ctr_Δ4.1 (n = 25); Cre_WT (n = 30); Cre_Δ4.1 (n = 27). **P < 0.01; ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, at least 3 independent experiments). (E) DIV8 to DIV10 WT mouse cortical neurons transfected with WT HA-Nrxn1α or HA-Nrxn1α Δ4.1 and treated with DMSO (vehicle) or Dynasore. Neurons were immunostained 18 h after treatment for surface HA-Nrxn1α (grayscale). (F) Quantification of panel E: ratio of axonal–dendritic surface HA fluorescence intensity. WT_DMSO (n = 40 neurons); Δ4.1_DMSO (n = 40); Δ4.1_Dynasore (n = 28). ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, at least 3 independent experiments). (G) HEK293T-cells expressing FLAG-Nlgn1 co-cultured with DIV10 Sorcs1^flox/flox cortical neurons infected with LV expressing mCherry (Control), Cre-T2A-mCherry, Cre-T2A-mCherry-T2A-WT Nrxn1α, Cre-T2A-mCherry-T2A-Nrxn1α Δ4.1, or Nrxn TKD; immunostained for FLAG (blue) and Synapsin1 (grayscale and green). (H) Quantification of panel G: the area of Synapsin1 clustering on the surface of Nlgn1-expressing HEK cells and normalized to cells expressing mCherry. Control (n = 29 neurons); Cre (n = 30); Cre-WT (n = 30); Cre-Δ4.1 (n = 30); TKD Nrxns (n = 30). *P < 0.05; ***P < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test, 3 independent experiments). (I) HEK293T-cells expressing NGL-3-EGFP co-cultured with DIV10 Sorcs1^flox/flox cortical neurons infected with LV expressing mCherry (Control) or Cre-T2A-mCherry and immunostained for EGFP (blue) and Synapsin1 (grayscale and green). (J) Quantification of panel I. Control (n = 30 neurons); Cre (n = 30). Underlying numerical values can be found in S1 Data. Graphs show mean ± SEM. Scale bars, 20 μm (panels A, C, and E); 5 μm (panels A [high-zoom], G, and I). Cre, Cre recombinase; Ctr, control; DIV, days in vitro; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEK293T, human embryonic kidney cells; LV, lentivirus; NGL-3, Netrin-G Ligand 3; Nrxn, neurexin; NS, nonsignificant; SorCS1, Sortilin-related CNS expressed 1; TKD, triple knock down; WT, wild type.