Fig. 3

SILAC combined with TRIM25 co-immunoprecipitation (co-IP) reveals a network of associated RNA-binding proteins. a A diagram of a co-IP experiment with T7-tagged TRIM25 in the presence of endogenous RNAs together with Gene Ontology enrichment map of proteins co-immunoprecipitated with T7-TRIM25. Enriched protein sets are represented as nodes (red circles) connected by edges (green lines), with the size of nodes and edges representing the number of proteins in gene sets and amount of overlap. b A diagram of a co-IP experiment with T7-tagged TRIM25ΔRBD in the presence of endogenous RNAs accompanied by the Gene Ontology enrichment map of proteins co-immunoprecipitated with T7-TRIM25ΔRBD. c A diagram of a co-IP experiment with T7-tagged TRIM25 in the presence of RNases together with Gene Ontology enrichment map of proteins co-immunoprecipitated with T7-TRIM25 in the presence of RNases. d Venn diagram representing numbers of overlapping proteins identified in the co-IPs with T7-TRIM25, T7-TRIM25ΔRBD, and T7-TRIM25 in the presence of RNases. e Validation of co-IP experiments show western blot against ZAP and DDX5 proteins. ZAP has two isoforms co-immunoprecipitated by T7-TRIM25 but not by T7-TRIM25ΔRBD or T7-TRIM25 in the presence of RNases. Note that anti-ZAP antibody is picking up a signal from overexpressed TRIM25. DDX5 binding to TRIM25 is partially RNA-dependent as co-IP by T7-TRIM25 in the presence of RNases decreases but does not eliminate its interaction with TRIM25. Lanes 1–3 represent loading controls. Lane 4 shows control co-IPs in mock HeLa cell extracts. Lanes 5–7 show anti-T7 co-IP. f Western blot analysis of wild-type (WT) and TRIM25 knock-out (KO) HeLa cells shows upregulation of ZAP protein in the KO cells. DDX5 levels do not change and serve as a loading control