Skip to main content
. 2024 May 15;15:4127. doi: 10.1038/s41467-024-48596-4

Fig. 5. Co-condensation with G3BP1 enhances ubiquitination activity of TRIM25 toward various AVPs.

Fig. 5

a Immunofluorescent staining of G3BP1 and K63-linked ubiquitin in TRIM25 KO HeLa cells re-expressing GFP-TRIM25 WT or ∆PTFG. Line scans show the related intensity profiles of GFP-TRIM25, G3BP1 and Ub. Bottom: Fluorescence intensities of and K63-linked ubiquitin in G3BP1-positive SGs. TRIM25 WT vs. TRIM25 ΔPTFG: p value: 1.89e−042; (b) WT or TRIM25 KO HEK293T WT cells were transfected with indicated GFP-TRIM25 constructs, and treated with or without poly(I:C). The cell lysates were subjected to immunoprecipitation with IgG or IGF2BP2 antibody followed by immunoblotting with indicated antibodies. c WT or G3BP1 KO HEK293T cells were transfected with the indicated plasmids and treated with or without poly(I:C). Anti-Flag immunoprecipitates were analyzed by immunoblot. d, e In vitro ubiquitination analysis of RIG-I-2CARD-HA and ZC3HAV1-N-HA. Assays contained E1 ligase, UbcH5b (E2 ligase), TRIM25 (E3 ligase), the 56-bp dsRNA, Ub, PBS, ITFG peptide (30 µM), and RIG-I-2CARD-HA (d) or ZC3HAV1-N-HA (e). Poly-Ub chain was analyzed by anti-Ub blot. Ubiquitination of RIG-I-2CARD-HA and ZC3HAV1-N-HA was analyzed by anti-HA blot. f Formation of 50 μM G3BP1 WT or mutant liquid droplets visualized by DIC microscopy. TBM TRIM25-bingding mutant, GDM G3BP1-dimmer mutant. Black arrows indicate the G3BP1 droplets. Quantitative analyses of G3BP1 droplet areas in (f). RIG-I-2CARD (g) and ZC3HAV1-N (h) in vitro ubiquitination assays with indicated G3BP1 mutants (10 μM) and E1/E2 (UbcH5b)/TRIM25. Assays contained purified E1 ligase, UbcH5b (E2 ligase), TRIM25 (E3 ligase), G3BP1 mutants (WT; TBM: TRIM25-binding mutant; GDM: G3BP1-dimmer mutant), the 56-bp dsRNA, Ub, RIG-I-2CARD-HA (d) or ZC3HAV1-N-HA (e). WT panels show corresponding control experiments, performed in parallel. Samples were analyzed by IB with anti-Ub and anti-HA. All data are representative of at least three independent experiments (a–h). Scale bars, 10 μm (a). (a.u. arbitrary units.) Mean ± s.e.m., statistical analysis was performed using two-tailed Student’s t test (a) or one-way ANOVA (f).