Figure 2
From: Cyclized NDGA modifies dynamic α-synuclein monomers preventing aggregation and toxicity
Interaction between NDGA and α-synuclein requires NDGA oxidation and cyclization. (A) NDGA treatment did not affect α-synuclein aggregation in the presence of N-acetylcysteine (NAC). α-Synuclein was aggregated for 3 days in the presence of 1:1 small molecules. After aggregation, PBS-insoluble α-synuclein was separated by centrifugation (21k g for 10 min). Soluble and insoluble fractions were boiled in SDS, run by SDS-PAGE, and colloidal stained. α-Synuclein in each fraction was quantified by in-gel densitometry. NAC was added at 20x molar excess to small molecule and catalase was added equal to 5% of protein, providing excess hydrogen peroxide decomposition capacity (n = 3–5, ***p < 0.001). (B) Colloidal staining of representative gels showed the formation of insoluble α-synuclein aggregates in the presence of NDGA and NAC. (C) Near-infrared fluorescent imaging of the same gels before colloidal staining showed a reduction of quinone-dependent fluorescence in the presence of NAC. (D) α-Synuclein did not aggregate in the presence of cyclizable analogs, NDGA and NDGA-5, but did with non-cyclizable NDGA-1 (n = 3, ***p < 0.001). (E) α-synuclein did not aggregate in the presence of cyclized cNDGA and cNDGA-5 (n = 3). α-Synuclein was aggregated for 3 days in the presence of 1:1 small molecules. After aggregation, PBS-insoluble α-synuclein was separated by centrifugation (21k g for 10 min). Soluble and insoluble fractions were boiled in SDS, run by SDS-PAGE, and colloidal stained. α-Synuclein in each fraction was quantified by in-gel densitometry.
