The Journal of Neuroscience, December 6, 2006, ():
Neuronal Pentraxin 1 Contributes to the Neuronal Damage Evoked by Amyloid-ß and Is Overexpressed in Dystrophic Neurites in Alzheimer's Brain
J. Neurosci. Abad et al.
26: 12735
Supplemental Data
Files in this Data Supplement:
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Supplemental Fig 1. Cortical neuronal cultures (4-6 DIV) were treated with Aβ 25-35. A) Time course of neuronal death induced by Aβ 25-35 (30 μΜ). Νeuronal viability was measured by PI fluorescence and expressed as a percentage of maximum neuronal death obtained with digitonin. B) Representative western blot showing the effect of Aβ 25-35 (30 μM) on NP1 over time. Western blots were incubated with mouse anti-NP1 antibody (1:1000). Actin was used as control for protein loading. C) Quantitative analysis of the effect of Aβ 25-35 on NP1 levels. Densitometric values of the bands representing NP1 immunoreactivity were normalized with the values of the corresponding actin bands. Values are the mean ± S.E.M of at least three independent experiments. *, significantly different from control values p<0.05, one-way ANOVA with Bonferroni post-hoc comparisons.
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Supplemental Fig 2. Specificity of anti-NP1 antibodies. The specifity of NP1 antibodies from different commercial sources was verified against recombinant NP1, NP2 and GFP proteins. Membranes were incubated with the following antibodies: From Beckton Dickinson (Los Angeles, CA): mouse anti-NP1 (1:1000), rabbit anti-NP1 (1:500). From Santa Cruz Biotechnology (Santa Cruz, CA): goat anti-NP1-C terminal (1:300), goat anti-NP1-N terminal (1:300), goat anti-NP2-C terminal (1:500), goat anti-NP2-N terminal (1:500). Peroxidase-conjugated secondary antibodies were: goat anti-mouse IgG (1:10000, Cell Signaling, Beverly, MA), goat anti-rabbit IgG (1:2000, Cell Signaling) and donkey anti-goat IgG (1:50000, Jackson Immunoresearch Laboratories, West Grove, PA). The results show that both mouse anti-NP1 and rabbit anti-NP1 from Becton Dickinson recognize specifically the recombinant NP1 protein. The anti-peptide antibodies from Santa-Cruz exhibit significant cross-reactivity between NP1 and NP2 recombinant proteins.
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Supplemental Fig 3. Silencing NP1 expression prevents the reduction of dendrite length evoked by Aβ. The effect of oligomeric Aβ 1-42 on dendrite length in primary cultures of cortical neurons was determined with immunofluorescence staining with the dendrite-specific monoclonal antibody MAP2 (2a+2b) (1:250, Sigma). The influence of NP1 was assessed by lentiviral-mediated RNA interference. Cortical neurons, plated at low density were transduced with the control lentivirus (pLVTHM-shRandom) or the lentivirus vector producing a NP1-specific siRNA (pLVTHM-shRNAi-NP1) at the same time of plating. Treatment with vehicle or oligomeric Aβ 1-42 (10 μM) was performed at 1 DIV for 72 h. The representative images show that treatment with oligomeric Aβ 1-42 reduces dendrite length. Silencing NP1 expression with shRNAi-NP1 prevents the effect of Aβ.
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Supplemental Fig 4. To verify the maturity or state of differentiation of our cultured neurons at the time of experimentation, we checked for the presence of dendrites and synaptic puncta at 5 days in vitro. We performed double-immunofluorescence and confocal microscopy studies with the dendrite-specific anti-MAP2 (2a+2b) monoclonal antibody (1:250, Sigma) and with the small vesicle associated protein anti-synapsin 1 polyclonal antibody (1:500, Sigma). The video shows that our cortical cultured neurons are polarized because MAP (2a+2b) staining (red) segregates in dendrites surrounded by synapsin clusters (green).
