Fig. 1.

Synapse density is proportional to eB3 expression. (A) DIV-8 (Upper) and DIV-21 (Lower) cortical neuron dendrites transfected with GFP at DIV 0 and stained with anti-eB3 (green), anti-PSD-95 (red), and anti-VGlut1 (blue) antibodies. Grayscale images show the pattern of eB3 staining. (B) Quantification of eB3 puncta density in neurons transfected with vector control (n = 27), eB3 shRNA#1 (n = 32), or eB3 shRNA#2 (n = 15). (C) DIV-9 neurons transfected with GFP plus indicated shRNA construct at DIV 0 and stained with anti-GFP (green), anti–PSD-95 (red), and anti-VGlut1 (blue) antibodies. Arrows indicate synaptic puncta identified by colocalized PSD-95 and VGlut1 immunostaining. (D) Quantification of colocalized PSD-95 and VGlut1 synaptic puncta density for vector control (n = 29), ephrin-B1 shRNA (n = 19), eB3 shRNA#1 (n = 31), or eB3 shRNA#2 (n = 16). (E) Quantification of colocalized PSD-95 and VGlut1 synaptic puncta density for vector control (n = 26), eB3 shRNA#1 (n = 23), or ephrin-B2 shRNA (n = 24). (F) DIV-9 cortical neuron dendrites transfected with GFP plus eB3 shRNA constructs and stained with anti-GFP (green), anti-eB3 (red), and anti-PSD-95 (blue) antibodies. Arrows and arrowheads indicate eB3 or PSD-95 puncta. (G and H) Plot of (G) eB3 puncta density or (H) eB3 staining intensity in arbitrary units vs. PSD-95 puncta density in DIV-9 neurons expressing GFP plus vector control [filled circles: (G) PSD-95 puncta density × eB3 puncta density, R² = 0.515, P < 0.0001; (H) PSD-95 puncta density × eB3 staining intensity, R² = 0.481, P < 0.0001] or eB3 shRNA#1 [open circles: (G) PSD-95 puncta density × eB3 puncta density, R² = 0.272, P < 0.002; (H) PSD-95 puncta density × eB3 staining intensity, R² = 0.354, P = 0.0002) stained with anti-eB3, anti–PSD-95, and anti-GFP antibodies. Plot is linear regression curve for all cells. (Scale bars, 3 μm.) Error bars indicate SEM. *P < 0.05, **P < 0.0001.