Extended Data Fig. 3: Liquid-liquid phase separation properties of OASL, RIPK3, and ZBP1.
a, Graph of putative intrinsically disordered regions of human OASL, RIPK3, and ZBP1 as calculated by PONDR (VSL2) algorithm. b, Failure of liquid droplet formation of purified mCherry-tagged full-length RIPK3 or C-terminal RIPK3295-518. Scale bar, 10 μm. c, Failure of liquid droplet formation of purified BFP-tagged full-length ZBP1. Scale bar, 10 μm. d, In vitro droplet formation by GFP-tagged full-length OASL, N-OAS, or C-UBL protein in a concentration-dependent manner. Incubation was carried out at physiological temperature and buffer for 1 h. Scale bar, 10 μm. b-d, Histogram represent distribution of quantified droplet amounts and diameters of the liquid droplets. e, Effect of KCl concentration on the formation of OASL liquid droplets. Scale bar, 10 μm. f, OASL-GFP droplets treated with increasing concentration of 1,6-hexanediol. Scale bar, 5 μm. g, Increase of Mg2+ concentration leads to enlargement of OASL droplets. Scale bar, 10 μm. h, In vitro liquid droplet formation of GFP-tagged mouse OASL (OASL1) treated with mock or poly(I:C) HMW (50 μg/ml) for 1 h. Scale bar, 10 μm. i, (Left) Schematic diagram of Sortase A-mediated labeling of FITC at the C-terminus of OASL with Sortase A-recognition motif (LPETGG). (Right) In vitro phase separation of FITC-labeled OASL treated with mock or poly(I:C) HMW (50 μg/ml). Scale bar, 10 μm. j,k, Representative FRAP images of OASL1 foci observed at (j) 4 h.p.i. and (l) 8 h.p.i. (l). SYTO 45-stained OASL1-Cherry foci were chosen for photobleaching. White rectangle box indicates the photobleached and recovered area within the foci. Scale bar, 10 μm. Data are representative of three (b-i) or two (j,k) independent experiments with similar results.
