Figure 2. Sema4D is sufficient to cluster presynaptic GABAergic components in the HEK293T cell—neuron coculture assay.
(A) Density of presynaptic GAD65 clusters formed in 9 DIV rat hippocampal neurons upon contact with a HEK293T cell expressing a specific cDNA, normalized to the density of the negative control condition. In this case, the negative control was HEK293T cells co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (B) Density of postsynaptic GABAARγ2 clusters formed in 9 DIV rat hippocampal neurons upon contact with a HEK293T cell expressing a specific cDNA, normalized to the density of the negative control condition. In this case, the negative control was HEK293T cells co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicated the control condition normalized to itself. (C) Representative images of GFP expressing HEK293T cells cocultured with 9 DIV rat hippocampal neurons immunostained for GABAergic synaptic markers, GAD65 (red) and GABAARγ2 (magenta). n > 101 cells per condition from ≥ 3 experiments/condition. *p < 0.05, Two-Way ANOVA with Tukey’s post hoc Test. Error bars are ± standard error of the average ratio. Scale bars, 10µm.