Fig. 2: WT and A53T-α-syn-Dendra2 show similar production and turnover rates even when insoluble pSer129 α-syn has accumulated in BSCs.

BSCs were transduced at 0 DIV with rAAVs to express Dendra2, WT-α-syn-Dendra2 or A53T-α-syn-Dendra2 and maintained in culture until 28 DIV. a BSCs were sequentially extracted to prepare soluble and triton-insoluble fractions. Representative western blots of the soluble and triton-insoluble fractions probed for pSer129 α-syn, total α-syn and β-actin as a loading control are shown. The mobility of molecular mass markers are shown on the left. Black arrow indicates pSer129 (81A), asterisks indicate non-specific bands. (n = 3). b Equal amounts of soluble and insoluble fractions were loaded onto Western blots and probed for total α-syn. Representative lanes are shown. The mobility of molecular mass markers are shown on the left. The proportion of insoluble α-syn of the total amount was calculated and presented on the bar graph (n = 4, mean ± SEM, analyzed by unpaired T-test). c BSCs were treated with 1 μM BI2536 or DMSO control for 24 h from 27-28 DIV to reduce levels of soluble pSer129. Representative blots of pSer129 α-syn (EP1536Y) and total α-syn are shown. The mobility of molecular mass markers are shown on the left. Bar chart shows levels of soluble pSer129 α-syn are reduced with BI2536 treatment but insoluble pSer129 α-syn is not (n = 3, data are mean ± SEM, analyzed by two-way ANOVA with post-hoc Bonferonni’s test). d BSCs were fixed, immunostained for pSer129 (EP1536Y) and confocal imaged to confirm the distribution of phosphorylated α-syn. Scale bar = 50 µm. (n = 3). e Schematic diagram shows the timeline of the long-term optical pulse labeling experiments using photoconversion of Dendra2, WT-α-syn-Dendra2 and A53T-α-syn-Dendra2 at 28 DIV when insoluble, phosphorylated α-syn has accumulated. f Representative images of photoconverted (red) and newly synthesized (green) Dendra2 above residual fluorescence in BSCs photoconverted at 28 DIV and imaged at several time points until 49 DIV. Merge of both channels is also shown. Scale bar = 50 µm g Line graph shows quantification of population levels of photoconverted Dendra2 fluorescent intensity in each condition that were quantified over time as a proportion of initial fluorescent intensity (n = 6, data are mean ± SEM, analyzed by Two-way ANOVA with post-hoc Sidak’s test). h Line graphs show quantification of population levels of green Dendra2 fluorescent intensity in each condition that were quantified over time as a proportion of initial fluorescent intensity (n = 6, data are mean ± SEM, analyzed by Two-way ANOVA with post-hoc Sidak’s test).