Figure 1. The intracellular C-terminus is not required for Sema4D to regulate GABAergic synapse formation.

(a) Diagram illustrating the epitope-tagged Sema4D and CD4 deletion and chimeric constructs used throughout this study. The myc epitope was inserted at the N-terminus while the FLAG epitope was inserted at the C-terminus of the peptides. The putative cleavage site between the Ig domain and transmembrane domain of Sema4D (Elhabazi et al 2001) is indicated by arrow.

(b) Quantification of inhibitory GABAergic synapse density at DIV14 as defined by overlapping GAD65/GABARγ2 puncta onto hippocampal neurons transfected with either an empty vector (“Control,” n= 47 neurons), an shRNA targeting Sema4D (“RNAi,” n= 35 neurons), overexpression of a myc tagged Sema4D RNAi resistant cDNA alone (“myc-Sema4D OE,” n=42 neurons), overexpression of a myc-tagged Sema4DΔC RNAi resistant cDNA alone (“myc-Sema4DΔC OE,” n=23 neurons), co-transfection of a the myc-Sema4D RNAi resistant cDNA along with an shRNA targeting Sema4D (“myc-Sema4D Rescue,” n= 43 neurons), or co-transfection of the myc-Sema4DΔC RNAi resistant cDNA along with an shRNA targeting Sema4D (“myc-Sema4DΔC Rescue,” n=31 neurons). Asterisk indicates p<0.05 using a univariate ANOVA compared to rescue conditions and control. Other pertinent p values include: p=0.665 control vs. myc-Sema4D rescue and p=0.244 control vs. myc-Sema4DΔC Rescue. Error bars denote standard error (SEM).

(c) (Left) Immunostaining against GAD65 (red) and GABARγ2 (blue) proteins on a stretch of GFP-positive dendrite from a representative neuron transfected with the constructs indicated on far right; overlapping puncta onto the transfected neuron appear white. (Right) GAD65 and GABARγ2 immunostaining in the absence of the GFP signal; overlapping puncta appear magenta. Scale bar =5um.