Figure 8.

Amino-Nogo-A resists to hydrogen peroxide (H₂O₂) via interacting with peroxiredoxin 2 (Prdx2). (a) Neurons were incubated with 0.2 μM HIV-1 trans-activating (TAT)-AM for indicated time, and then cells lysates were performed to western blot with anti-Prdx2-SO3 and anti-2-cysteine (Cys) Prdx2 antibodies. β-Actin was selected as a loading control. (b) Lysates from neurons treated by H₂O₂ with or without TAT-AM were performed to western blot with anti-Prdx2-SO3 and anti-2-Cys Prdx2. β-Actin was selected as a loading control. (c) Cortical neurons were incubated with 0.2 μM TAT-GFP or TAT-AM for 2 h, and then were submitted to Ni²⁺ pull-down assay. Bound endogenous Prdx2-SO3 was analyzed by immunoblotting with anti-Prdx2-SO3 antibody. (d) Glutathione-Sepharose beads coupled with GST or GST-AM were incubated with TAT-HA-Prdx2. Bound Prdx2 was analyzed by immunoblotting with anti-HA antibody. (e) Glutathione-Sepharose beads coupled with GST or GST-Prdx2 were incubated with 293FT cell lysate overexpressed with GFP-M1-WT. Bound M1-WT was analyzed by immunoblotting with anti-GFP antibody. The amount of GST or GST-fusion Prdx2 was revealed by naphthol blue black staining. Neurons pretreated with TAT-Prdx2 or TAT-AM, followed by incubation in H₂O₂ was examined by cell death assay (f) and dihydroethidium (DHE) test (g) as before. (h) Top: neurons transfected with ctrl-small interfering RNA (siRNA) and Prdx2-siRNA for 48 h were blotted with anti-2-Cys Prdx2 antibody. β-Actin was selected as a loading control. Lower: Neurons transfected with ctrl-siRNA and Prdx2-siRNA were pre-treated with TAT-AM followed by incubation in H₂O₂, and then cell death assay was calculated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (i) Top: neurons treated with 0.2 μM TAT-M1-WT, TAT-M1-3CA and TAT-M1-2CA for 2 h were lysed and immunoblotted with anti-HA antibody. The effect of the three proteins on oxidative damaged neurons was checked by cell death assay (lower in i) and DHE test (j). (k) In all, 100 ng M9-WT pre-incubated with 1 mM H₂O₂ or not were labeled with biotin-conjugated iodoacetamide (BIAM) and then probed with horseradish peroxidase (HRP)-streptavidin. (l) Working model of amino-Nogo-A resisting to H₂O₂. n=4, mean±S.D., one-way analysis of variance (ANOVA), ^**P<0.01; ^***P<0.001