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DVDY_23874_sm_SuppFig1.tif8219KSupporting Information Figure 1. Blocking of ephrin-A5 induced growth cone turning by unclustered EphA4/Fc recombinant protein. Ephrin-A5/Fc protein or IgG Fc protein clustered with Cy3-conjugated anti-Fc was laid down in stripes on poly-ornithine-coated glass by the Bonhoeffer method (Nature Protocols 2:1216). Hippocampal neurons were isolated from perinatal mice (Nature Protocols 2:1490) were plated on top of the stripes in the presence of soluble EphA4/Fc or IgG Fc (as control) and allowed to grow for 48 hr. Cells were fixed and stained with an antibody to acetylated tubulin to visualize neurites. A: Ten images as shown here were taken of each group. A blinded observer scored each axon in each panel for turning or stopping when it encountered a red stripe, and determined a % turning score for each group. B: Frequency of neurite turning represented as fold difference relative to that observed on control IgG Fc stripes for ephrin-A5/Fc stripes and ephrin-A5/Fc stripes incubated with unclustered EphA4/Fc protein. The EphA4 protein effectively blocked the neurite turning activity of ephrin-A5 that would normally stop or turn away axonal growth from hippocampal neurons (J Neurosci 20:8885).

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