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Supplemental Figure 1: Nrp1/Plexin-A complexes do not mediate Sema3Ainduced FAK recruitment (A) Sema3A does not induce FAK recruitment to Nrp1/Plex-A2 and Nrp1/Plex-A3 complexes. (B) In Nrp1/Plex-A4-expressing cells immunoprecipitation of either FAK or Plex-A4 did not reveal Plex-A4/FAK interaction, both in cont and Sema3A-treated condition. LpIP: Lysate post IP. (D) Immunodetection of L1 forms mutated on serine residues, L1S1194L and L1S1224L expressed in COS7 cells showing that L1 proteins are present at the cell surface. The ser mutations do not prevent L1/Nrp1 coprecipitation. Supplemental Figure 2: FAK and L1 signaling are required for the guidance of cortical axons in slice overlay assays. Schematic drawing and microphotographs of the assay to illustrate the quantification of axon trajectory of cortical neurons grown on top of cortical slices. Axons are normally directed away from the pial surface by the secretion of Sema3A. As shown by the histogram, over-expression of L1S1194 and FAFKD strongly alter the trajectory taken by axons from neurons over-expressing L1WT and FAKWT. Supplemental figure 3: Defects of cortical projections in horizontal sections from mice lacking Plexin-A3, Plexin-A4 or L1. (A) Schematic diagram of horizontal brain sections of neonatal brain showing the position of the corpus callosum and the internal capsule. (B) Immunolabelling of horizontal brain sections illustrating the reduced density of Nrp1-expressing axons in the intermediate zone (black arrows) and extending from lateral cortical regions in the internal capsule (black asterisks) of Plexin-A3/A4 -/-, Plexin-A3- and Plexin-A4-/mice, compared with wild-types. Note that the decrease of Nrp1-expresising axons found in the double mutant is fully reproduced in the simple Plexin-A4 null mutant while is also present but less pronounced in the simple Plexin-A3 null mutant. The reduction of fibers in the internal capsule can also be visualized by antibodies to L1. (C) Immunodetection of Nrp1 in horizontal brain sections illustrating the sharp reduction of Nrp1+ axons in the internal capsule of mice lacking L1, compared to wild-type mice. DCC labeling shows abnormal fasciculation of axons still present in the internal capsule of mice lacking L1. Scale bar: 1 mm. CP: cortical plate; FT: fiber tract; IC: internal capsule. (C) DCC labelling of callosal axons shows that the corpus callosum is formed normally in Plexin-A3/A4 mice. Supplemental Figure 4: Antero-posterior organization of Nrp1+ axons in the corpus callosum of Plexin-A3, Plexin-A4 and L1 knockout mice. Nrp1+ axons are positioned at the anterior pole of the corpus callosum, as illustrated by these horizontal sections immunolabelled with antibodies to Nrp1. This organization is maintained in mice lacking Plexin-A3 and Plexin-A4. In contrast in mice lacking L1, sections were found for which Nrp1+ axons were no longer present. Scale bar: 1 mm. CC: Corpus callosum