Extended Data Fig. 1: IRF3 undergoes LLPS.
a, Domain structure (upper) and the intrinsically disordered tendency (lower) of IRF3. IUPred2, ANCHOR2 and VSL2 assigned scores of disordered tendencies between 0 and 1 to the sequences were shown. b, Bacterial purified GFP-IRF3 wt, d_DBD, d_IDR, d_IAD and d_ID proteins were analyzed by SDS-PAGE and detected by Coomasssie blue staining. c–e, 5 μM GFP-IRF3 were treated with 5% Hex (c), heated-inactivated (5 min at 95 °C and immediately put on ice for 5 min) (d), or treated with 100 μg/ml Proteinase K for 30 min at 40 °C (e) and then subjected to droplet formation assay in vitro (200 mM NaCl, pH 7.0, room temperature). Mean ± s.d., n = 6 independent experiments. f, 3D reconstruction of activated GFP-IRF3 puncta in HeLa cells followed by stimulation with SeV for 12 h. Series z-stack images of live cells were captured by confocal microscope and then 3D reconstruction was performed. Typical optical sections of z-stack were shown. g, Left: a schematic describing the generation of site-specific phosphorylated recombinant IRF3 protein with a Sep-accepting tRNA (tRNASep) and its cognate phosphoseryl-tRNA synthase (SepRS), which incorporates the phosphorylated serine on the amber codon. Right: IB of the purified protein with antibody specific to phospho-Ser386 and phospho-Ser396 of IRF3. The anti-Myc blot indicates loading of lanes. h, Immunofluorescence microscopy and DAPI staining of L929 cells showed nuclear puncta of endogenous IRF3 upon PBS or SeV stimulation for 12 h. i, Related to Fig. 2e: quantified percentages of cells harboring GFP puncta and GFP nuclear puncta upon SeV stimulation were shown. n = 3. j, qPCR analysis of IFNB1 mRNA in IRF3 KO cells transfected with indicated plasmid (s) followed by mock infected (PBS) or infection for 12 h with SeV. n = 3. Data are representative of three independent experiments (b, f, g, h–j). n = 6 biological independent samples (c–e). Scale bar, 5 μm (c–f, h). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–e, i, j).
