Extended Data Fig. 2: R3 is necessary for RIG-I LLPS.
From: Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing
a, The pi–pi contacts of R1, R2 and R3 were determined using PScore (https://pound.med.utoronto.ca/~JFKlab/Software/psp.htm). The assessment of NCPR (net charge per residue), FCR (fraction of charged residue), hydrophobicity, and Shannon Entropy of R1, R2 and R3 was conducted with localCIDER (https://pappulab.github.io/localCIDER/). The displayed score is the phase separation prediction score. Features, from top to bottom, are pi interaction, NCPR, FCR, hydrophobicity, and Shannon Entropy. b, Related to Fig. 2a, immunoblot (IB) analysis of the indicated proteins; FL, full length. c, Related to Fig. 2f, percentage of cells with puncta was shown; n = 3. d, Representative micrographs of droplet formation (left) and quantification (right) of 5′ triphosphate double-stranded RNA (5′ppp-dsRNA, 1 μM) with purified GFP-RIG-I WT (3 μM) or GFP-RIG-I R3 where R3 was replaced by GGS linker (3 μM); n = 3. e, Immunofluorescence and DAPI staining of HeLa cells transfected with GFP-RIG-I WT and R3GGS followed by stimulated with SeV for 12 h. Quantification of cells with GFP-RIG-I puncta was shown (right); n = 3. Data are representative of at least three independent experiments. Scale bar, 10 μm (e), 20 μm (d). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–e); ****P < 0.0001. Exact P values, source numerical data and unprocessed blots are provided.
