Figure 1.
The affinity and specificity of the mAbs to NogoA proteins were determined using Western blot and IHC staining.
A: The membrane was blotted with different dilutions (1∶500, 1∶5000, 1∶20000) of anti-NogoA pAb, aNogo66 mAb, and aNogoA-N mAb. The corresponding bands were near 200 kDa. B: The aNogo66 mAb and aNogoA-N mAb recognised NogoA expressed in oligodendrocytes in the white matter of the spinal cord thoracolumbar coronary segment. Colocalisation (yellow), as indicated by arrowheads, of anti-NogoA pAb (red) with aNogo66 mAb (green) or aNogoA-N mAb (green). C: Colocalisation (yellow), as indicated by arrowheads, of anti-MBP mAb (red) with aNogo66 mAb (green) or aNogoA-N mAb (green). D: Absence of colocalisation of anti-GFAP mAb (red) with aNogo66 mAb (green) or aNogoA-N mAb (green). Scale bars = 50 µm.
Figure 2.
Identification of the epitopes recognised by the two mAbs by Western blot.
A: Several peptide epitopes from NogoA were produced. A library of six Nogo deletion constructs was made, and GST-tagged recombinant proteins were designed. B: Lysates of induced BL21 bacteria with recombinant plasmids were analysed using Coomassie brilliant blue staining (lanes from left to right: lysates of induced BL21 bacteria with recombinant plasmids GST-ΔNogoA-Na, GST-ΔNogoA-Nb, GST-ΔNogoA-Nc, GST-ΔNogoA-Nd, GST-ΔNogo66a, and GST-ΔNogo66b; lane 7: lysate of induced BL21 bacteria with empty pGEX-4T-1 vector; lane 8: lysates of non-induced BL21 bacteria alone). C: Recombinant proteins identified by WB. Anti-GST antibody was used to detect the fragments (lanes from left to right: the recombinant proteins GST-ΔNogoA-Na, GST-ΔNogoA-Nb, GST-ΔNogoA-Nc, GST-ΔNogoA-Nd, GST-ΔNogo66a, and GST-ΔNogo66b; lane 7: GST protein; lane 8: non-induced). D: The epitope recognised by the aNogoA-N mAb (lanes from left to right: the recombinant proteins GST-ΔNogoA-Na, GST-ΔNogoA-Nb, GST-ΔNogoA-Nc, GST-ΔNogoA-Nd and GST protein). E: The epitope recognised by the aNogo66 mAb (lanes from left to right: the recombinant proteins GST-ΔNogo66a and GST-ΔNogo66b and GST protein).
Figure 3.
aNogo66 mAb and aNogoA-N mAb enhanced the axon growth of hippocampal neurons by blocking NogoA inhibition.
A: Dissociated rat E18.5 hippocampal neurons were cultured on the control substrate PLL (control group). B, D: Hippocampal neurons cultured on 100 pmol NogoA FC (aa 1026–1090) or NogoA FC (aa 544–725) exhibited strongly inhibited axon growth. C, E: Hippocampal neurons were cultured on NogoA FC (aa 1026–1090) and treated with aNogo66 mAb, or neurons were cultured on NogoA FC (aa 544–725) and treated with aNogoA-N mAb, to assess the contribution of the two mAbs on inhibition. F: Statistical analysis of hippocampal neuron axon growth in each group is expressed as the mean ± SEM of each group in each separate experiment (##P<0.01, b group or d group vs. a group; **P<0.01, c group vs. b group and e group vs. d group. n = 6 wells per condition; scale bars = 100 µm).
Figure 4.
The aNogo66 mAb and aNogoA-N mAb enhanced branch formation and suppressed myelin inhibition.
A, C: The function of aNogo66 mAb or aNogoA-N mAb on branch formation against NogoA was assessed by immunofluorescence. The arrowheads indicate the axon branch points (n = 6 wells per condition; scale bars = 50 µm). B: For statistical analysis, the number of axon branch points per neuron is represented as the mean ± SEM from one representative experiment (*P<0.05, b group vs. a group; **P<0.01, d group vs. c group). D: For statistical analysis, the distance that axons sent out their first branches from the cell body was expressed as the mean ± SEM from one representative experiment (**P<0.01, b group vs. a group; d group vs. c group).
Figure 5.
aNogo66 mAb and aNogoA-N mAb reduce the inhibition exerted by the targeted Nogo-A region on axon outgrowth and branching.
A: Dissociated rat E18.5 hippocampal neurons were cultured on the control substrate PLL (Normal group). B, C, D: Hippocampal neurons cultured on 100 pmol NogoA FC (aa 1026–1090); E, F, G: Hippocampal neurons cultured on 100 pmol NogoA FC (aa 544–725); C, F: Treated with aNogo66 mAb; D, G: treated with aNogoA-N mAb. I: Statistical analysis of the axon growth is expressed as the mean ± SEM of each group in each separate experiment. J: Statistical analysis of the number of axonal branch points is expressed as the mean ± SEM of each group in each separate experiment (**P<0.01, C group vs. B or D group; **P<0.01, G group vs. E or F group; *P<0.05, C group vs. B or D group; *P<0.05, G group vs. E or F group; scale bars = 100 µm)
Figure 6.
Western blot analysis of the levels of GAP43 expression on the fifth and seventh days after primary hippocampal neuron culture in vitro.
(n = 6 wells per condition). A: Hippocampal neurons were grown on 100 pmol NogoA FC (aa 1026–1090) substrate for the control group; hippocampal neurons were grown on NogoA FC (aa 1026–1090) and treated with aNogo66 mAb for the Nogo66 mAb group; B: Data are expressed as the level of the GAP43-positive band in the control group and the Nogo66 mAb group on the fifth day or seventh day. *P<0.05. C: Hippocampal neurons grown on the NogoA FC (aa 544–725) substrate were the control group; hippocampal neurons grown on NogoA FC (aa 544–725) and treated with aNogoA-N mAb were the aNogoA-N mAb group; D: Data are expressed as the level of GAP43-positive substance in the control group and the aNogoA-N mAb group on the fifth day or seventh day. *P<0.05.