Fig. 8.

tPA and plasminogen bind the NG2 present in injured spinal cord. tPA (A) and plasminogen (B) were incubated with injured spinal cord homogenates in a solid-phase binding assay and adherent NG2 was detected. Data represent three separate experiments for each time point (n = 2). (C) Recombinant NG2 and tPA were used to generate a binding curve. (D) NG2 was immunoprecipitated from injured spinal cord homogenates, and analyzed using SDS-PAGE zymography to assess the presence of active tPA in the immunoprecipitated complex. tPA (64 kDa; black arrow on the right side of the gel). Beads alone (beads) or beads incubated with preimmune serum (Beads + serum) were used as negative controls. TH, total homogenates; IP, immunoprecipitated material. The lower band of activity is most likely uPA. In the presence of the inhibitor amiloride, the band disappears. (E) NG2 immunoprecipitated material was analyzed by western blot using a rat anti-mouse NG2 antibody. NG2 Ctr, NG2 detected in total homogenates; NG2 IP, NG2 detected in immunoprecipitated material. Beads alone (beads) or beads incubated with preimmune serum (Beads + serum) were used as negative controls. (F) Representative western blot showing the upregulation of NG2 in Day 7 SCI lysates from wild-type (wt) or tPA^−/− animals (using the rat anti-mouse NG2 antibody) compared to noninjured control levels. Arrows point to NG2 bands. Error bars represent S.E.M where *P < 0.05; **P < 0.001 by ANOVA followed by Bonferroni’s post hoc test. (G, H) Quantification of NG2 levels from western blots of wt and tPA^−/− not-injured animals, and animals 7 days after SCI (n = 3 experiments with groups varying between 4 and 6 animals). Results were plotted as ratios of NG2 levels in each sample over those in not injured animals for each genotype and analyzed by ANOVA followed by Bonferroni’s post hoc test (P = 0.061). Similar trends were evident 5 and 14 days post SCI. Samples were normalized for total protein levels using plasminogen levels as control protein (data not shown).