Skip to main content
. Author manuscript; available in PMC: 2021 Nov 20.
Published in final edited form as: Cell Rep. 2021 Oct 19;37(3):109828. doi: 10.1016/j.celrep.2021.109828

Figure 3. Robo2 induces excitatory synapse formation in a Slit-dependent manner.

Figure 3.

(A) In vitro hemi-synapse assay (24h coculture of primary neurons at DIV12 with HEK293 cells expressing cDNAs as indicated in text boxes on the left for each row of images). Immunostaining for Vglut1 indicates presence of excitatory presynaptic boutons, which cluster around the perimeter of the HEK293 cell in case the cDNA is synaptogenic. NLG1: positive control, CD8: negative control. Robo1 and Robo2 expression in HEK293 cells leads to Vglut1 clustering around the cell perimeter. Slit-binding deficient receptors, Robo3 and Robo2ΔIg1,2, Robo2[L93P], are unable to induce Vglut1 clustering. Scale bar: 7μm.

(B) Robo2 does not induce inhibitory synapse formation as evidence by the absence of clustering the inhibitory presynaptic marker Vgat1 in immunohistochemistry. Scale bar: 7 μm.

(C and D) Quantification of hemi-synapse assay. F: Fluorescence intensity in AU (arbitrary units). Statistics: one-way ANOVA, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, whiskers show min/max, n = 4 independent experiments with at least 20 cells/coverslip (triplicates of one condition per independent experiment).