Subcellular fractionation and IRF3 dimerization assay

Subcellular fractionation was performed for HEK293T or MEFs as described¹⁴. In brief, cellular lysate in hypotonic buffer (Buffer A: Tris-Cl 10 mM pH 7.5, KCl 10 mM, EGTA 0.5 mM and MgCl₂ 1.5 mM) was obtained and centrifuged at 1,000 g for 5 min. The supernatant (S1) was isolated and further centrifuged at 10,000 g for 10 min. The supernatant (S5) and pellet (P5) were then separated. S5 was subjected to centrifugation at 100,000g for 30 min, and the resultant supernatant (S100) was collected. P5 was treated with RIG-I and Ub mixture as indicated, which was then isolated and incubated with S5 in the presence of ATP, followed by native gel electrophoresis to examine IRF3 dimerization as described¹⁴.