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. 2026 Feb 6;12(6):eadz3483. doi: 10.1126/sciadv.adz3483

Fig. 1. Cytosolic peptide library and degron screening.

Fig. 1.

(A) The peptide library consists of 214,158 different 30-residue partially overlapping fragments covering 5672 cytosolic human proteins. Parts of the figure were created in BioRender. R. Hartmann-Petersen (2025), https://BioRender.com/23ydezu. (B) Schematic illustration of the expression and screening system. The plasmid containing the GFP-fused peptide library also includes an internal ribosomal entry site (IRES), mCherry, and a site for Bxb1 recombination into a landing pad in HEK293T cells. Expression from the landing pad is regulated by the Tet-on promoter (bent arrow), which, in nonrecombined cells, 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 (2A). Upon correct integration, the GFP fragments and mCherry are expressed from the same mRNA. Using fluorescence-activated cell sorting (FACS), cells are sorted into four equally populated bins, and the fragments in each bin can be identified by sequencing. Parts of the figure were created in BioRender. R. Hartmann-Petersen (2025), https://BioRender.com/qw20nnx and adapted from (17, 43, 47). Representative distributions of GFP:mCherry ratios in cells expressing the library and either untreated (control) or (C) treated for 8 hours with cycloheximide (CHX; 10 μg/ml) (n = 900,000), (D) for 16 hours with 15 μM bortezomib (BZ) (n = 720,000), or (E) 16 hours with 1 μM MLN7243 (n = 347,077; untreated, n = 340,000). (F) A representative flow cytometry (FC) profile for cells expressing the library (n = 495,000). Bin thresholds used to sort the library into four (bin 1 to bin 4) equally populated bins (25% in each bin) are shown as black horizontal bars.