Figure 2.

Pretreatment of HIV-1 trans-activating (TAT)-amino-Nogo-A protects cortical neurons against hydrogen peroxide (H₂O₂)-induced cell death. (a) Schematic presentation of the Nogo proteins. Human Nogo-A, amino acids (aa) 1–1192; Nogo-B, aa 1–373; and Nogo-C, aa 1–199. N terminus aa 1–185 is shared by Nogo-A and Nogo-B; C terminus aa 188 is the reticulon homology domain within Nogo-A/B/C and other reticulon family members. AN, the full-length amino-Nogo containing 1–1004 residues; AM, the 186–1004 residues unique to Nogo-A. (b) Top: pTAT-HA-AN/AM expression vector. 6 × His, 6 His domain mediates protein binding to Ni²⁺ column; TAT, HIV-1-trans-activating short peptide helps protein enter cells; HA, a tag inserted for subsequent protein detection. AN/AM, insertion of desired cDNA using the restriction enzymes. Lower: neuron lysates treated with PBS, TAT-GFP, TAT-AN and TAT-AM for 2 h were blotted using anti-HA antibody. Corresponding bands at 150, 130 and 36 kDa represented TAT-AN, TAT-AM and TAT-GFP, respectively, and the black rectangle represented the nonspecific bands of anti-HA antibody. (c) Under phase-contrast microscopy, numerous neurons exposed to 50 μM H₂O₂ for 12 h exhibited damaged signs, disappeared cell body with brightening and protruding nuclei and discontinuous neurites. However, neurons with TAT-AN or TAT-AM pretreatment remained as normal. (d) The percentage of cell death rate was calculated by propidium iodide (PI) (+)/Hoechst (+). (e) Cell death rate was also examined by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (f) The cultured neurons were pre-incubated with TAT-AM at 0, 0.02, 0.05, 0.2 and 0.4 μM for 2 h, followed with H₂O₂ treatment. Cell death rate was calculated through PI (+)/Hoechst (+). (g) TAT-AM (0.2 μM) was added to the neuron cultures before 2 h, simultaneously or post 5 or 30 min of H₂O₂ treatment. Cell death rate was calculated through PI (+)/Hoechst (+). Bar=50 μm. n=4, mean±S.D., one-way analysis of variance (ANOVA), ^*P<0.05; ^***P<0.001