Af1494

Neutralization experiments using specific blocking antibodies were conducted to determine which neurotrophic factors were involved in mediating PC12 cell survival and neurite outgrowth. PC12 was cultured with 50% DPSCs-CM in the presence of anti-NGF antibody (0.25 µg/mL; Cat# MAB556, R&D Systems), anti-GDNF antibody (1 µg/mL; Cat# AF212, R&D Systems), anti-BDNF antibody (2 µg/mL; Cat# AF1494, R&D Systems), anti-NT-3 antibody (2 µg/mL; Cat# AF1404, R&D Systems) or in the presence of mixture of all antibodies using the concentrations stated earlier.
At the beginning of each experiment, cells were washed twice with PBS and all experiments were performed in absence of serum. The cultures were maintained under the same conditions as previous experiments for 8 days, followed by quantification of neurite outgrowth and the number of survived neurons.

Spinal cords were extruded with ice-cold PBS (pH 7.4), 6 days after injury, and the dorsal horn of the lumbar enlargement corresponding to L3 to L5 was isolated. Following BDNF injections, spinal cords were extruded with ice-cold PBS (pH 7.4) at times 0 (no injection), 3, 6, and 24 hours, and the entire lower lumbar enlargement was used for WB. The tissue was lysed in standard Tris-NaCI-EDTA lysis buffer supplemented with protease and phosphatase inhibitors. Following SDS–polyacrylamide gel electrophoresis (PAGE) and blotting, the proteins were probed against NTSR-3 (1:1000; 61200, BD Biosciences), KCC2 (1:1000; 07-432, Millipore), TrkB (1:1000; AF1494, R&D Systems), ERK1/2 (1:1000; 4695, Cell Signaling Technology), pERK1/2 (1:1000; 9101, Cell Signaling Technology), Akt (1:500; 9272, Cell Signaling Technology), pAkt (1:300, 9275, Cell Signaling Technology), NKCC1 (1:1000; Lytle, C., Developmental Studies Hybridoma Bank, NICHD, The University of Iowa), pNKCC1 (1:1000; donated by B. Forbush, Yale University, New Haven, CT), and β-actin (1:5000; Sigma-Aldrich), visualized with horseradish peroxidase–conjugated secondary antibody (Dako) and ECL substrate (Amersham) by LAS 4000 (Fujifilm Life Science) and quantified using Multi Gauge version 3.2 (Fujifilm Life Science).