1) Generation of dopaminergic neurons: Patient skin fibroblasts carrying the p.V15A variant were reprogrammed into iPSCs using Sendai virus to deliver the four reprogramming factors OCT4, SOX2, KLF4, and cMYC (CytoTune-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher Scientific). One iPSC clone was established according to the manufacturer’s protocol and cultured on Matrigel-coated dishes (BD Biosciences) in mTeSR1 medium (STEMCELL Technologies). The line was characterized for the expression of pluripotency markers and spontaneous differentiation potential to form embryoid bodies as described previously (PMID: 30316041). A normal karyotype as well as the absence of large chromosomal aberrations was confirmed by single polymorphic nucleotide (SNP) profiling. Genome integrity was assessed by Illumina GSA-24v3.0 beadchip array and analyzed with GenomeStudio software (Illumina). iPSC lines from four control individuals (SFC084-03 (https://hpscreg.eu/cell-line/STBCi033-B), SFC086-03 (https://hpscreg.eu/cell-line/STBCi052-C), SFC089-03 (https://hpscreg.eu/cell-line/STBCi053-A), SFC156-03 (https://hpscreg.eu/cell-line/STBCi101-A) were generated and characterized previously (PMID: 28827786). The direct differentiation of iPSCs into dopaminergic neurons was conducted as described before (PMID: 28379402 ). Neurons were harvested at day 86-100 of differentiation. 2) Native Dot Blot Analysis: Total protein was extracted from cell pellets using RIPA Lysis buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% DOC, 1% NP-40, 0.1% SDS, and proteinase inhibitor cocktail). Protein extracts were used for the DC Protein Assay (Bio-Rad), and 7.5 μg of total protein extracts per sample were subjected to native dot blot analyses as previously described (PMID: 35722765). Total protein staining Revert 700 Stain Solution (Li-COR) was used as a loading control. Antibody signal intensities were normalized to the total protein. Antibodies used for the blotting analysis were as follows: primary antibody MJFR-14-6-4-2 (1:2000 #ab209538; Abcam) overnight at 4°C and secondary antibody IRDye 800CW Goat anti-Rabbit (1:20000; Li-COR) for 1h at room temperature. Detection and digitalization were performed using Odyssey CLx Infrared Imaging System (Li-COR). All the native dot blots originated from the same experiment and were processed in parallel.
