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. 2024 Feb 20;15:1541. doi: 10.1038/s41467-024-45829-4

Fig. 1. Assessment of Parkin variant abundance.

Fig. 1

A Schematic illustration of VAMP-seq applied to a site-saturated library of PRKN variants. The PRKN plasmid DNA library (Vector DNA) comprises a Bxb1-specific recombination site, a PRKN variant fused to GFP, an internal ribosomal entry site (IRES), mCherry and a unique barcode. The barcoded PRKN library is introduced into HEK293T cells containing a landing pad locus. The landing pad contains a Bxb1 recombination site downstream of a Tet-on promotor (bent arrow) that drives the expression of BFP, inducible Caspase 9 (iCasp9) and a blasticidin resistance gene (BlastR) separated with a parechovirus 2A-like translational stop-start sequence (2 A). The cells are co-transfected with the barcoded PRKN library and a plasmid encoding the Bxb1 recombinase that catalyzes site-specific recombination. After correct integration, GFP-Parkin and mCherry are expressed from the same mRNA. Using fluorescence-activated cell sorting (FACS) cells are sorted into four different and equally populated bins and the Parkin variants in each bin can be identified by sequencing the barcode. Figure created with BioRender.com. B Live fluorescence microscopy images showing BFP, GFP and mCherry signal intensities in stable transfected landing pad cells expressing N-terminal GFP-tagged wild-type (WT) or low abundance variant R42P Parkin. C The protein levels of GFP-tagged Parkin in whole cell lysates from stable transfected landing pad cell lines expressing N- or C-terminally GFP-tagged wild-type (WT) or R42P Parkin determined using SDS-PAGE and Western blotting with an antibody against GFP. The protein level of mCherry and GAPDH served as a control for cell-to-cell fluctuations and as a loading control, respectively. D Representative flow cytometry scatter plots of landing pad cells expressing GFP-WT (blue, n = 52,023), GFP-R42P (red, n = 52,245) or the PRKN variant library (grey, n = 5.46×105). E Representative histogram plots for stable transfected landing pad cells expressing N-terminal GFP-tagged wild-type Parkin (GFP-WT) or R42P (GFP-R42P). Each histogram was created from at least 46,500 cells. F A representative flow cytometry profile for landing pad cells expressing the PRKN library (grey, n = 4.95×105). Bin thresholds used to sort the library into four (1-4) equally populated bins (25% in each bin) are shown by black horizontal range gates.