Figure 1.
Aβ 1–42 oligomer distribution in N2a cells.
A) High magnified, fluorescence microscopy pictures showing the cellular localization of fluorescent dye 6 hours after 30 µM Aβ 1–42-EDANS large oligomers incubation. The fluorescence is mainly confined to the cytoplasm [C] and did not penetrate into the nucleus [N]. Scale bar 10 and 5 µm. B). The kinetic of accumulation was carried out by time lapse recording experiments coupled to fluorescence microscope acquisition (40× of magnification). Each single image represents the merge between the contrast phase signal and the fluorescence of the field excited in the UV range (from 380 to 425 nm of wavelength), Scale bar 40 µm. C-D-E) N2a cells were treated with 30 µM Aβ 1–42 large oligomers for 6 hours prior to immunocytochemistry analysis. C) vimentin, D) GRP-78 and E) cathepsin D (all FITC) plus Aβ 6E10 staining (TRIC 546); nuclei were stained with Hoechst 33285. Scale bar. 15 µm. Images were merged by superimposing single fluorescence images.
Figure 2.
Far Western Blot with cytosolic (C) and membrane proteins (M).
Fifteen µg of proteins from cytosol and membrane fractions were blotted after electrophoresis. Far Western Blot was performed with A) 100 ng/ml of untagged Aβ 1–42 enriched in oligomers. Actin is shown as a marker for the cytosolic fraction. B) 100 ng/ml of EDANS-tagged Aβ 1–42 enriched in oligomers.
Table 1.
Identification of membrane proteins able to bind Aβ 1–42 large oligomers.
Figure 3.
Aβ toxicity and Far Western Blot assays.
A) N2a cell viability after treatment with Aβ 1–40 species or Aβ 1–42 species, concentration range from 0.22 µM to 10 µM and 50 nM to 5 µM respectively. ANOVA followed by Bonferroni's post hoc test; *** p<0.001; * p<0.05; p>0.05 not significant (ns). Mean and SD; N = 3 or more different experiments, 4 replicates each. B-C-D-E) Comparisons of binding capacities among different peptide preparations. Fifteen µg proteins from cytosolic (cyt) and membrane fractions (mem) were run on SDS-PAGE and blotted onto nitrocellulose. Far Western Blot was performed with B 1) 100 ng/ml Aβ 1–42 oligomers; B 2) 100 ng/ml Aβ 1–40 monomers; C 1) 100 ng/ml Aβ 1–42 monomers; C 2) 100 ng/ml Aβ 1–42 oligomers; D1) 100 ng/ml Aβ 1–42 oligomers; D2) 100 ng/ml Aβ 1–42 fibrils; E1) 1 µg/ml Aβ 1–40 monomers; E2) 1 µg/ml Aβ 1–40 fibrils; E3) 1 µg/ml Aβ 1–40 oligomers. Membranes were incubated with peptide solutions separately; then pooled to be stained concurrently with the same antibody incubations and the same film exposure.