Journal: Cell Death and Differentiation

Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

doi: 10.1038/cdd.2011.206

Figure Lengend Snippet: Nogo-A expression in cortical neurons. (a) The diagram for antibodies against different antigen recognition sites of human Nogo-A NiR-Δ2 (amino acids (aa) 57–185), NiG-Δ20 (aa 564–749) and Nogo-66 (aa 1055–1120) are the three inhibitory regions of human Nogo-A. TMs are transmembrane domains located on the two sides of Nogo-66. The arrows indicate the target regions of five Nogo-A-specific antibodies, polyclonal antibody (pAb) A201 against aa 201–250, monoclonal antibody (mAb) A563 against aa 563–627, mAb 1E8 against aa 570–691 and pAb A620 against aa 620–1004. 2B3 is targeted to Nogo-66, aa 1055–1120. (b) Top: the expression of neuronal Nogo-A was immunostained with A620 and TuJ1, an immature neuronal marker. Lower: subcellular distribution of neuronal Nogo-A was blotted with A620. Cytosolic (F1), organelle/membrane (F2), nucleic (F3) and cytoskeleton fractions (F4) were the four subcellular fractions of neurons. (c) Living cell staining for neurons were performed with pAb A620, mAb 1E8 or pAb A201 (top). The A620 and 1E8 antibodies had previously been shown to label the extracellular amino-Nogo-A in oligodendrocytes (lower). (d) The total RNA was extracted from neurons exposed to 50 μM hydrogen peroxide (H2O2) for 8 h or not and submitted to quantitative real-time polymerase chain reaction (PCR). β-Actin was selected as an inner standard. (e) Cell lysates from neurons exposed to 50 μM H2O2 for 8 h or not were blotted with pAb A620. β-Actin was selected as an inner standard (left). Quantification by densitometric scans was presented by Nogo-A/β-actin (right). Bar=50 μm. n=3, mean±S.D., paired t-test, *P<0.05, ***P<0.001

Article Snippet: 7 , 15 For western blots, A620 and two commercial antibodies, A201 (Prosci, San Diego, CA, USA; no. 4089) and A563 (BD Biosciences PharMingen, San Diego, CA, USA; no. 612238) were used.

Techniques: Expressing, Marker, Membrane, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Death and Differentiation

Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

doi: 10.1038/cdd.2011.206

Figure Lengend Snippet: In all, 290–562 residues are the pivotal domain of amino-Nogo-A for resisting to oxidative damage. (a) Schematic diagram of different deletions (M1–M9) of amino-Nogo. (b) Neurons were pretreated with 0.2 μM HIV-1 trans-activating (TAT)-M1-M9 for 2 h, followed by exposure to 50 μM hydrogen peroxide (H2O2) for another 12 h in the presence of TAT-M1-M9. Cell death rate was calculated by propidium iodide (PI) (+)/Hoechst (+). (c) Lysates from neurons treated by H2O2 with or without TAT-AM was performed to western blot with anti-HA and A201 antibodies. β-Actin was selected as a loading control. *Presented the nonspecific bands of anti-HA antibody. (d) HEK293FT transfected with mock or full-length human Nogo-A were treated with indicated concentrations of H2O2 for 1 h, and cell lysates were subjected to western blot probed with A201 antibody. β-Actin was selected as a loading control. The 48 kDa bands were pointed with arrow; n=4, mean±S.D., one-way analysis of variance (ANOVA), *P<0.05; ***P<0.001

Article Snippet: 7 , 15 For western blots, A620 and two commercial antibodies, A201 (Prosci, San Diego, CA, USA; no. 4089) and A563 (BD Biosciences PharMingen, San Diego, CA, USA; no. 612238) were used.

Techniques: Western Blot, Control, Transfection