Fig. 5: Ngb oxygen-binding site controls p38 binding/activation and axon regeneration in neurons upon OGD/Re. | Cell Death & Disease

Fig. 5: Ngb oxygen-binding site controls p38 binding/activation and axon regeneration in neurons upon OGD/Re.

From: Neuroglobin boosts axon regeneration during ischemic reperfusion via p38 binding and activation depending on oxygen signal

Fig. 5

a Effects of Ngb knockdown on neurite regeneration in N2a cells during hypoxia. N2a cells stably overexpressing shNgb or N-con were subjected to 1% O2 incubation for various times. Representative micrograph and statistical analysis demonstrated that Ngb knockdown suppressed neurite regeneration in N2a cells during hypoxia. Data are presented as means ± S.E.M. **P < 0.01 and ***P < 0.001, N = 3. b Effects of Ngb overexpression on Ngb neurite regeneration in N2a cells during hypoxia. N2a cells were transiently transfected with pEGFP-N1-Ngb or pEGFP-N1 for 24 h and then subjected to 1% O2 incubation for various times. Representative micrograph and statistical analysis demonstrated that Ngb overexpression promoted neurite regeneration in N2a cells at 6 h of hypoxia. Data are presented as means ± S.E.M. ***P < 0.001, N = 3. c Effects of Ngb oxygen-binding site mutations on p38 interaction in neuronal cells upon OGD/Re. N2a cells were co-transfected with pGST-p38 and pEGFP-N1-Ngb mutants. Results of GST pull-down assays showed that only NgbH64L mutant (increasing O2-binding affinity) evidently increased p38-binding ability. d Effects of Ngb oxygen-binding site mutations on p38 activation in neurons upon OGD/Re. Cultured neurons were subjected to lentiviral infection with Venus-Ngb mutant-LV or Venus-LV at DIV 2 and subjected to OGD-1 h/Re-24 h at DIV 7. Results of western blotting analysis showed that NgbH64A (reducing O2-binding affinity) evidently reduced p-p38 in neurons after OGD/Re. e Effects of Ngb oxygen-binding site mutation on axon regeneration in the neurons after OGD/Re. Cultured neurons were subjected to lentiviral infection with Venus-Ngb mutant-LV and subjected to OGD-1 h/Re-24 h. Statistical analyses demonstrated that mean axon length was significantly increased after NgbH64L overexpression and that Ngb-induced axon regeneration was abolished by p38 MAPK inhibitor. Data are presented as means ± S.E.M. **P < 0.01, N = 3

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