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. 2021 Oct 1;12:5772. doi: 10.1038/s41467-021-26061-w

Fig. 1. Identification of RNF213 as ISG15-binding protein.

Fig. 1

a Workflow to map ISG15 interaction partners. Virotrap and Glutathione-S-Transferase (GST) pull-down were employed as two orthogonal methods. In Virotrap, GAG-ISG15 was expressed in HEK293T cells. Twenty four hours post-transfection, cells were treated with interferon (IFN)-α for 24 h of left untreated. Budding virus-like particles (VLPs) containing ISG15 and its interaction partners were purified, lysed and digested into peptides prior to LC-MS/MS analysis. In GST pull-down, glutathione beads were decorated with GST-ISG15 and mixed with a cellular lysate from HEK293T, HeLa, or THP-1 cells. Prior to lysis cells were treated for 24 h with interferon-α (HEK293T) or interferon-β (HeLa and THP-1). Following on-bead digestion, the resulting peptides were analyzed by LC-MS/MS. b Volcano plot showing the result of a t-test to compare VLPs containing mature ISG15 versus VLPs containing dihydrofolate reductase from Escherichia coli (eDHFR) as negative control (n = 4 replicates). Proteins outside the curved lines represent specific ISG15 interaction partners. Proteins identified as ISG15 interaction partners in all virotrap screens are annotated (n = 29) and listed in Supplementary Data 1. c VLPs containing ISG15 or eDHFR were collected and analyzed by immunoblot (IB) against RNF213, ISG15, and GAG. FLAG-RNF213 purified from a lysate of HEK293T cells was loaded as a positive control and confirmed the presence of an RNF213 band in the ISG15 VLPs. d–f Volcano plots comparing GST pulldowns using GST-ISG15-coated beads with GST-coated beads as control in lysates of HEK293T, THP-1, and HeLa cells (n = 3). Proteins significantly enriched in PDs with ISG15-coated beads are annotated. g GST PDs with ISG15, ubiquitin, and SUMO followed by immunoblotting show binding of RNF213 to ISG15, but not to ubiquitin or SUMO. Beads coated with each UBL were mixed with a lysate of HEK293T cells expressing FLAG-RNF213 and bound proteins were analyzed by immunoblot against FLAG and GST. h, i Validation of GST PD assays with ubiquitin and SUMO using RNF31 and RNF4 as known binders of these UBLs, respectively. Assays were performed as in g. Source data are provided as a Source Data file.