Fig. 1: αSyn-dependent accumulation of FluoReSyn in HEK293 cells.

a Schematic representation of the initial observation of cells transiently expressing NbSyn87-EGFP alone (left) or together with hαSyn (right). Although the former group of cells showed minimal fluorescence, the latter presented with a strong EGFP signal. b Alignment of the amino-acid sequence from hαSyn and Rpn10 across different species. Rpn10 residues similar to hαSyn in the putative epitope are displayed in red. c Schematic representation of the proposed mechanism of degradation versus stabilization of SynNb87-EGFP in the presence or absence of hαSyn. The following Protein Data Bank (PDB) accession numbers were used and modified to assemble the schematic: 2Y0G (EGFP), 2 × 6 M (Nanobody), 1XQ8 (hαSyn), 6MSK (Proteasome). d Schemes of constructs used to transfect HEK293 cells (left). For the stably transfected cells, a tetracycline-inducible promotor (TetON) was used, followed by a mCherry reporter sequence, a cleavable T2A sequence, and FluoReSyn made of NbSyn87, EGFP and a nuclear localization signal (NLS) sequence. Transient expression of untagged wild-type human hαSyn was driven by a plasmid containing a cytomegalovirus (CMV) promotor. Equally scaled, representative images of doxycycline-induced Reporter-cells (right). Cells were either mock transfected (control) or transiently transfected with the hαSyn expression constructs. Scale bar represents 10 µm e Quantitative analysis of EGFP-positive cells transfected with variable quantities of hαSyn plasmid. Per replication and condition >1000 cells were analyzed. Error bars represent the SEM from three independent experiments (n = 3). f Western blot analysis of lysates from Reporter-cells transfected with variable quantities of hαSyn. Immunoblotted (IB) anti-EGFP represents FluoReSyn. Loading control is IB Beta-Actin. Full length blots are displayed in Supplementary Fig. 1d. Source data is available as a Source data file.