Fig. 4: Designed binders bind to their targets in cells and binder G3BP1-11 modulates G3BP1 phase separation.
From: Diffusing protein binders to intrinsically disordered proteins
a, Colocalization of IL-2RG-30, G3BP1-11 and BRCA1_ARATH-35 with their full-length targets in HeLa cells. Scale bars, 10 µm (top row) and 20 µm (middle and bottom rows). b, Colocalization of amylin-22, amylin-36, amylin-68n, VP48-2 and CP-35 with their targets. Scale bars, 20 µm. c, The LC–MS/MS recovery percent of amylin from phosphate-buffered saline (PBS)–0.1% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) buffer and EDTA-anticoagulated plasma was compared between BSA-blocked tosyl-activated bead, an off-target binder, and the binder amylin-68n. Percent recovery was calculated relative to the peak area of pure amylin in elution solvent (100% recovery). The data indicate mean ± s.d. (n = 3 technical replicates). d,e, Phase diagrams showing effects of G3BP1-11 (d) and control (e) on G3BP1–RNA phase separation across 0–8 ng μl−1 RNA. Red indicates phase-separated droplets, and blue indicates mixed states. f,g, Without binder, confocal images indicate that G3BP1 forms cytoplasmic puncta after 1 h of arsenite treatment (f). Quantification of G3BP1 puncta per cell (each point represents one cell; n = 20 cells per condition), showing a notable increase upon arsenite treatment, is also displayed (g). h,i, With the G3BP1-11 binder, arsenite treatment failed to induce a similar increase in puncta. Confocal images (h) show colocalization of G3BP1 (green) and the co-expressed mScarlet-tagged binder (red). Quantification revealed no markedly increase in puncta after arsenite treatment (i). Scale bars, 10 μm (f,h).
