GPR56 and interacting proteins are enriched in pseudopods after induction by membranes of DRG neurons dependent on BDNF and NT-3. (A) Schematic representation of the pseudopod assay. DRG were dissected from E15.5 rats. Neurons were selected with BDNF and NT-3 growth factors. After purification, neurons were collected and homogenized. SCs were induced or not with neuronal membrane suspension. Pseudopods (Ps) were isolated from cell bodies (CBs), and both were analyzed by MS. (B) Quantification by bicinchoninic acid assay of the protein content of SC pseudopods and CBs after induction. Data are shown as mean ± SD; n (independent experiment) = 3. *, P < 0.05. (C) Distribution of proteins enriched by neuronal membranes. The abscissa axis indicates relative polarization of a protein between P and CB. The ordinate axis indicates relative enrichment of a protein by neuronal membranes in comparison to noninduced condition. Both GPR56 and GNA13 are found enriched in SC Ps after neuronal membranes induction. (D) Protein ontology analysis of the pathways most enriched in SC Ps. RhoA signaling is enriched by neuronal membranes. For more complete listings, see Dataset 1. Student’s t test (unpaired) was used to test for statistical significance. (E) GTP-RhoA pull-down assay revealed a significant decrease in the proportion of GTP-RhoA (active) to total RhoA in Gpr56^−/− and Gpr56^+/− animals relative to WT controls on P6 (**, P < 0.01, unpaired Student’s t test; data are shown as mean ± SEM; n = 4 nerves from n = 2 animals per technical replicate; n = 3 technical replicates).