Figure 3.

Alpha-synuclein and phospho-synuclein DNA binding is increased by the minor groove-binding agent Hoechst 33258 and competes with the major groove-binding agent methyl green.A, increasing concentrations of the DNA minor groove binder Hoechst 33258 (0–16 μM) does not change the signal generated by DNA (left side of each gel) but does increase shifted fraction caused by 14 μM alpha-synuclein (aSyn) and serine-129 phosphorylated alpha-synuclein (pSyn; aSyn Hoechst EC50 = 21.29 μM, R² = 0.653, deviation from zero slope p = 0.0003, three-parameter dose–response curve; N = 4 gels; pSyn Hoechst EC50 = 12.76 μM, R² = 0.395, deviation from zero slope p = 0.0002, x-axis Hoechst concentration on log scale). B, in contrast, high concentrations (at 500 nM and above) of the DNA major groove binder methyl green decrease intercalation and/or fluorescence of the Sybr dye used to image DNA (left side of aSyn and pSyn gels). Addition of 29 μM aSyn or pSyn competes with methyl green and partially restores Sybr dye fluorescence signal. Methyl green does not affect the shifted fraction of DNA in the presence of 29 μM aSyn or pSyn at values where it does not reduce Sybr dye fluorescence (at 250 nM and less, aSyn: R² = 0.205, deviation from zero slope p = 0.5473; pSyn: R² = 0.748, deviation from zero slope p = 0.1351; N = 3 gels, x-axis methyl green concentration on log scale). The 14 and 29 μM aSyn and pSyn concentrations were chosen since these produced intermediate levels of shift, allowing for detection of possible changes with small-molecule dye addition.