Figure 6. Spinal Cord Injury-Specific Induction of ProNGF.

(A) High-molecular weight NGF is predominantly induced after spinal cord injury. 30 μg of extracts were analyzed at each time point in Western analyses for NGF (Chemicon), BDNF (Promega), and NT3 (Promega). The extracts were prepared from rostral 5 mm (rats) or 3 mm (mice) blocks from the lesion center, but the pattern of NGF expression did not vary whether the sample was taken caudally or from the lesion center. The arrow indicates the position of recombinant BDNF and NT3, which were used at 250 ng as a control for antibody specificity. The star points to purified NGF being weakly recognized by anti-BDNF antibody.

(B) The high-molecular weight NGF is proNGF. 350 μg of lysates was immunoprecipitated with proNGF antibody, and probed with anti-mature-NGF antibody (Chemicon). The bands at 26-32 KDa disappear almost completely following depletion with proNGF antibody, indicating that these bands are proNGF (compare lanes dep and undep). Although not shown, mature NGF is present in undepleted rat samples in longer exposure.

(C) Specificity of proNGF and proBDNF antibodies. Western blot of 50 ng of mature NGF (Harlan Bioproducts for Science), 50 ng of mature BDNF (Promega), 50 μg of lysate from 293 cells stably expressing cleavage resistant proNGF, and 250 ng of supernatant from 293 cells infected with cleavage-resistant adenoviral proBDNF. A set of pro and mature NGF was probed with anti-pro/mature NGF antibody (Cedarlane) and proNGF antibody, and a set of pro and mature BDNF was probed with anti-mature-BDNF (Santa Cruz) and anti-proBDNF antibody.

(D) ProNGF antibody depletes proNGF, but not mature NGF. Recombinant proNGF (lanes 1 and 2) or recombinant pro and mature NGF (lanes 3 and 4) were subjected to immunodepletion using proNGF antibody. Note that proNGF antibody depletes only proNGF, but not mature NGF (compare lanes 3 and 4).