Fig. 10. The RNF213 E3 module is required for its antimicrobial activity.

a Glutathione-S-Transferase (GST) pull-down with ISG15 followed by immunoblotting shows that 3xFLAG-RNF213ΔC binds to ISG15 similar to full-length 3xFLAG-RNF213. Beads coated with GST-ISG15 were mixed with a lysate of HEK293T cells expressing full-length 3xFLAG-RNF213, 3xFLAG-RNF213ΔC, or the complementary C-terminal fragment of RNF213 and bound proteins were analyzed by immunoblot (IB) against FLAG and GST. b FLAG immunoprecipitation (IP) was performed from lysates of HEK293T cells expressing 3xFLAG-RNF213, 3xFLAG-RNF213ΔC, or FLAG-eGFP as control. After immunoprecipitation, beads were mixed with a lysate of HEK293T cells expressing HA-ISG15 and the ISGylation machinery (E1, E2, E3). 3xFLAG-RNF213ΔC was capable of pulling down ISGylated proteins similar to 3xFLAG-RNF213, while FLAG-eGFP was not. c HeLa cells were infected with Listeria monocytogenes EGD for 4 h at MOI 25. Twenty four hours prior to infection, HeLa cells were transfected with plasmids encoding 3xFLAG-RNF213 or 3xFLAG-RNF213ΔC or with an empty vector (mock) as control. Intracellular Listeria were quantified after serial dilution by counting colony-forming units (CFUs) in a gentamycin assay. The percentage of intracellular bacteria relative to mock plasmid-transfected cells is shown (lower panel, AVG ± SEM, n = 3 independent experiments, two-tailed unpaired t-test, n.s. not significant). Immunoblots against FLAG with tubulin as loading control confirmed expression of FLAG-RNF213, FLAG-RNF213ΔC (upper panel). d Representative images of HeLa cells transfected with eGFP-RNF213ΔC and infected for 18 h with Listeria monocytogenes EGD stably expressing mCherry. Scale bars in the pictures and insets are respectively 10 microns and 0.5 micron. eGFP-RNF213ΔC showed a diffused cellular staining, not decorating intracellular Listeria (DAPI = 4′,6-diamidino-2-phenylindole). In c asterisks indicate p values with ****p < 0.0001. Source data are provided as a Source Data file.