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Fig. 3 | Molecular Brain

Fig. 3

From: The small GTPase ARF6 regulates GABAergic synapse development

Fig. 3

ARF6 activity is required for GABAergic synapse development in cultured neurons. a Cultured hippocampal neurons were transfected with a lentiviral vector expressing sh-Control, sh-Arf6, or coexpressing sh-Arf6 and shRNA-resistant ARF6 expression vectors (ARF6-WT, ARF6-T27 N, or ARF6-T157A) at DIV8 and analyzed at DIV14 by double-immunofluorescence staining with antibodies to EGFP (blue) and the indicated synaptic markers (GAD67, gephyrin, or GABAARγ2). b Summary data showing the effects of ARF6 KD on synaptic puncta density (left) and synaptic puncta size (right), measured using GAD67, gephyrin, and GABAARγ2 as synaptic markers. More than two dendrites per transfected neuron were analyzed and group-averaged. Data are presented as means ± SEMs from three independent experiments (n = 12–18 neurons; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; non-parametric ANOVA with Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test). c Cultured hippocampal neurons were transfected with a lentiviral vector expressing sh-Control, sh-Arf6, or coexpressing sh-Arf6 and shRNA-resistant ARF6 expression vectors (ARF6-WT, ARF6-T27 N, or ARF6-T157A) at DIV8 and analyzed at DIV14 by double-immunofluorescence staining with antibodies to EGFP (blue) and surface GABAARγ2 (red). d Summary data showing the effects of ARF6 KD on the density of surface GABAARγ2+ puncta (left) and size of surface GABAARγ2+ puncta (right). More than two dendrites per transfected neuron were analyzed and group-averaged. Data are presented as means ± SEMs from three independent experiments (n = 12–18 neurons; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; non-parametric ANOVA with Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test). e Cultured hippocampal neurons were transfected with a lentiviral vector expressing sh-Control, sh-Arf6, or coexpressing sh-Arf6 and shRNA-resistant ARF6 expression vectors (ARF6-WT, ARF6-T27 N, or ARF6-T157A) at DIV8 and analyzed at DIV14 by triple-immunofluorescence staining with antibodies to EGFP (blue), VGAT (red) and gephyrin (green). f Summary data showing the effects of ARF6 KD on the colocalized puncta density of VGAT and gephyrin (left) and size of colocalized puncta (right). More than two dendrites per transfected neuron were analyzed and group-averaged. Data are presented as means ± SEMs from three independent experiments (n = 16 neurons; ***p < 0.001 vs. control; non-parametric ANOVA with Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test)

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