Figure 4

Repellent axon guidance is disrupted in the presence of an anti-proBDNF antibody or a soluble extracellular domain of p75^NTR. (A) Quantification of axon growth preferences in the presence of an anti-proBDNF antibody [14] or a control antibody. Stripe assay experiments were performed in the presence of a proBDNF antibody (1:200) [14] or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody (see also Additional file 4). Error bars represent the standard error of the mean. Statistics were performed using Kruskal-Wallis test and Dunn's multiple comparison test with ***P < 0.001. (B) Immunoprecipitation and western blot analysis of CHO cell supernatants transfected with p75^{NTR, sol, FLAG} analysing two different clones (c7 and c9). The arrowhead points to the band corresponding to p75^{NTR, sol, FLAG}; the asterisk indicates the Ig light chain from the αFLAG antibody. The first lane shows the analysis of mock-transfected cells. IB, immunoblot; IP, immunoprecipitation. (C-F) Single cells from E6 retina were electroporated with an eGFP expression plasmid and plated on alternating lanes of EphA7-Fc/Fc, or Fc/Fc (see (A)). Two days later, the cultures were fixed and analysed for their growth preferences. (C, E) Fc versus Fc stripes. (D, F) EphA7-Fc versus Fc stripes. (C, D) Addition of media from mock transfected CHO cells. (E, F) Addition of media from CHO cells transfected with an expression plasmid for the extracellular soluble domain of p75^NTR (p75^{NTR, sol}). (G) Quantification of axon growth preferences in the presence of p75^{NTR, sol} or control medium. Two different p75^{NTR, sol, FLAG} clones (c7 and c9) were analysed and led to a similar reduction in the growth preference of RGC axons. Statistics were performed in GraphPad using Kruskal-Wallis with post hoc Dunn's multiple comparison test.