Fig. 4. Co-localization of Esyt1 at synaptic level after AAV8-hSyn-DIO-hEsyt1 and AAV-phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE administration in En1^cre and SOD1^G93A;En1^cre mice.

a Cartoon depicting experimental approach with dual virus injection and Proximity ligation assay (PLA). AAV8-hSyn-DIO-hEsyt1 and AAV-phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE were injected at 1:1 concentration. PLA recognizing Esyt1 and Gfp proteins was used to validate Esyt1 expression at synaptic level. b Lower magnification microphotograph shows Gfp targeting also other cells in the intermediate area of the spinal cord (GFP in green, PLA in red, Nissl in blue). c–e Negative control experiment without primary antibody incubation does not show PLA signal on motor neurons (d). f–h Three weeks after viral injection, and overexpression, Esyt1 is located synaptically on motor neurons in En1^cre and (i–k) SOD1^G93A;En1^cre mice. PLA reveals interaction of Esyt1 and Gfp antibodies at synaptic level after combined viral delivery of (in red, g, j), that co-localizes with Gfp signal alone (in green, h, k). Findings were replicated in two SOD1^G93A;En1^cre and two En1^cre mice. Images (c–k) are acquired at 63x magnification. Arrows indicate co-localization. Scale bar in c = 5 μm. Source data are provided as a Source Data file.