Fig. 3. USP18-deletion exacerbates the cellular ISGylated protein network.

a Immunoblot using specific antibodies against the indicated proteins showing additional ISGylation of these proteins in the HAP1 USP18 KO cells treated with IFN. b HAP1 USP18 KO cells were transfected with siRNA sequences targeting ISG15 and ADAR and treated with IFN for the indicated times. After ISG15 silencing, modified ADAR (ADAR1 (p150)-ISG15; lower panel) is no longer visible by immunoblot. c HAP1 WT and USP18 KO cells were treated with IFN for the indicated times, lysed and subjected to immunoprecipitation (IP) with ISG15 antibodies. Enriched ISGylated proteins were present in eluates from the USP18 KO cells (top panel) but there was no enrichment for ubiquitylated proteins (lower panel). d Immunoblot of the same eluates in (c) against the indicated proteins showing enrichment for ISGylated species. e Volcano plot of comparative proteomic analysis of the IP eluates in (c, d). Pathways enriched are shown. Comparative scatter plot (f) of the GlyGly peptide IP and the ISG15 IP, performed in the same experimental conditions showing a strong overlap between the two data sets and strong enrichment of members of the ISG cancer gene signature⁵ and factors upregulated in PD1-responders.⁴³