Inhibition of proteasomal degradation lowers αsyn solubility.

<p>(<b>A</b>) Quantitative analysis of GFP fluorescence of cells expressing αsyn-GFP₁₁+ GFP_1–10 (blue) and TP αsyn-GFP₁₁+ GFP_1–10 (red). Relative fluorescence was calculated by normalizing the fluorescence of cells 12 and 24 hrs post transfection to the fluorescence measured at 0 hr. *<i>p</i><0.005; **<i>p</i><0.05. (<b>B</b>) Relative fluorescence of cells expressing αsyn-GFP₁₁ and GFP_1–10 (blue) and TP αsyn-GFP₁₁+ GFP_1–10 (red), 24 hrs post transfection. Cells were incubated for 24 hrs with increasing concentrations of lactacystin (0–5 µM). Relative fluorescence was evaluated by normalizing the fluorescence of treated cells to the fluorescence of untreated cells. *<i>p</i><0.01, **<i>p</i><0.05. Data points are reported as mean ± S.E.M. (n = 3) (<b>C</b>) Representative western blot of cells expressing αsyn-GFP₁₁, treated with lactacystin (5 µM) for 24 hrs, using αsyn-specific antibody. (<b>D</b>) Western blots band quantification of cells expressing αsyn-GFP₁₁. Bands were quantified by NIH ImageJ analysis software. GAPDH was used as loading control.</p

ProteoStat® co-localization assay.

a<p>Low co-localization – 35 to 60, yellow pixels.</p>b<p>High co-localization – 0 to 35, red pixels.</p

Inhibition of proteasomal degradation enhances αsyn aggregation.

<p>Immunofluorescence microscopy of cells expressing (<b>A</b>) αsyn-GFP₁₁ and GFP_1–10 and (<b>B</b>) TP αsyn-GFP₁₁ and GFP_1–10. Cells were treated with lactacystin (5 µM) for 24 hrs. Co-localization intensity of αsyn (blue, column 1) and ProteoStat® dye (red, column 2) is displayed in the form of co-localization heat maps (column 3). Hot colors represent positive co-localization and cold colors represent negative co-localization. Scale bars represent 10 µm.</p

The αsyn-split GFP system enables quantification of soluble αsyn in HeLa cells.

<p>HeLa cells were transfected with αsyn-GFP₁₁ and GFP_1–10. Fluorescence was measured with a flow cytometer and live cells were imaged using fluorescence microscopy. (<b>A</b>) Representative fluorescence histogram (left panel) and data analysis (right panel) of cells expressing GFP_1–10 only. Over 99% of the cell population does not display fluorescence. A representative image of live cells is reported in the figure inset. (<b>B</b>) Fluorescence histogram (left panel) and data analysis (right panel) of cells co-expressing αsyn-GFP₁₁ and GFP_1–10. Over 50% of the cell population display GFP fluorescence. A representative fluorescence microscopy image is reported in the figure inset. Scale bars represent 200 µm.</p

Mutations in αsyn gene sequence affect protein solubility.

<p>The fluorescence of cells expressing the αsyn-split GFP system was measured by flow cytometry and fluorescence microscopy. (<b>A</b>) Quantitative analysis of GFP fluorescence of HeLa cells expressing αsyn-GFP₁₁+ GFP_1–10 (blue), A53T αsyn-GFP₁₁+ GFP_1–10 (green), αsyn123-GFP₁₁+ GFP_1–10 (purple) and TP αsyn-GFP₁₁+ GFP_1–10 (red). (<b>B</b>) Quantitative analysis of GFP fluorescence of H4 cells expressing αsyn-GFP₁₁ (blue), A53T αsyn-GFP₁₁+ GFP_1–10 (green), αsyn123-GFP₁₁+ GFP_1–10 (purple) and TP αsyn-GFP₁₁+ GFP_1–10 (red). Fluorescence measurements were normalized to fluorescence of cells expressing wild type αsyn-GFP₁₁. *<i>p</i><0.05; **<i>p</i><0.005. Data points are reported as mean ± S.E.M. (n = 3). (<b>C</b>–<b>D</b>) Representative fluorescence microscopy images of HeLa cells expressing αsyn-GFP₁₁+ GFP_1–10 (blue, first row), A53T αsyn-GFP₁₁+ GFP_1–10 (green, second row), αsyn123-GFP₁₁+ GFP_1–10 (purple, third row) and TP αsyn-GFP₁₁+ GFP_1–10 (red, fourth row) at 20X (C) and 100X (D) magnification. GFP fluorescence is shown in the left column and mCherry fluorescence is shown in the right column. Scale bar represents 200 µm (C) and 20 µm (D).</p