Fig. 4: Ngb 7–122 aa fragment is required for p38 binding and activation upon OGD/Re.

a Representative results of BiFC showed the direct binding of Ngb and p38 in living N2a cells. N2a cells were co-transfected with NV-p38+CV-Ngb/CV or CV-p38+NV-Ngb/NV plasmids at a ratio of 1:1 and subjected to OGD/Re after 24 h of transfection. Recombinant Venus signal was visualized under a fluorescent microscope. b Statistical analysis of the relative recombinant Venus signal. Mean fluorescence intensity from nine fields of each culture was calculated and values >1.5-fold of the background were considered positive. Data are presented as means ± S.E.M. ***P < 0.001, N = 3. c Schematic structure of Ngb showing the cutting sites for Ngb truncates. d Interaction of Ngb truncates and p38. N2a cells were co-transfected with pEGFP-N1-Ngb truncates and pGST-p38 plasmids at a ratio of 1:1 and subjected to OGD-1 h/Re-24 h. Equal amount of proteins were subjected to GST pull-down and western blotting analysis. Ngb truncates or p38 was detected with anti-GFP or anti-GST antibodies correspondingly. e Effects of Ngb truncates on p38 activation in N2a cells after OGD/Re. Representative western blotting results showed that the deletion of Ngb 1–18 but not 1–6 amino acids evidently reduced p-p38 expression in N2a cells after OGD-1 h/Re-24 h. f Effects of p38 MAPK inhibitor on Ngb-induced neurite regrowth in N2a cells after OGD/Re. N2a cells were transfected with p-ENGF-N1-GFP or vector and subjected to OGD-1 h/Re-24 h. p38 MAPK inhibitor SB203580 was supplemented during reoxygenation incubation. Mean neurite length of the longest neurite of over 100 N2a cells was used for statistical analysis. Data are presented as means ± S.E.M. *P < 0.05, N = 3