Extended Data Fig. 2: IRF7 undergoes LLPS with IRF3.
a, Domain structure (upper) and the intrinsically disordered tendency (lower) of IRF7. IUPred2, ANCHOR2 and VSL2 assigned scores of disordered tendencies between 0 and 1 to the sequences were shown. b, Bacterial purified mCherry-IRF7 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 mCherry-IRF7 were treated with 5% Hex (c), 100 μg/ml Proteinase K for 30 min at 40 °C (d), or treated with heated-inactivated (5 min at 95 °C and immediately put on ice for 5 min) (e), and then subjected to droplet formation assay in vitro (200 mM NaCl, pH 7.0, room temperature). f, Representative fluorescence and DIC images of mCherry-IRF7 (5 μM) droplets formation at room temperature with indicated concentrations of NaCl at pH 7.0. g, Representative fluorescence and DIC images of mCherry-IRF7 (5 μM) droplets formation at indicated temperature with 200 mM NaCl at pH 7.0. Scale bar, 10 μm. h, Left: representative micrographs of mCherry-IRF7 (5 μM) droplets before and after photobleaching. Right: quantification of FRAP of mCherry-IRF7 droplet over a 200 s time course (mean ± s.d., n = 3 droplets). i, Time-lapse micrographs of the fusion of AF594 labeled IRF7 (10 μM) droplets at room temperature with 200 mM NaCl at pH 7.0. n = 3. j, Purified mCherry-IRF7 wt, d_DBD, d_IDR, d_IAD, and d_ID proteins (5 μM) was analyzed using droplet formation assays at room temperature with 200 mM NaCl at pH 7.0. k, Fusion upon contact of droplets formed by GFP-IRF3 and mCherry-IRF7 proteins. l, Related to Fig. 3k: the endogenous IRF7 puncta were quantified in control and IRF3 KO cells. Data are representative of three independent experiments (b, i, k). n = 6 biological independent samples (c–g, j, l). Scale bar, 5 μm (c–k). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–g, j, l).
