Extended Data Figure 4. ISG15 controls the stability of the USP18 protein, but not of other interferon-stimulated-gene products.

a, HLLR1-1.4 cells were transfected with either control siRNA or ISG15 siRNA. One day post-transfection, cells were stimulated with IFN-β (500 pM) for 6 h. Cycloheximide (CHX, 20 µgml⁻¹) was then added for various time periods, from 30 min to 5 h. Cell lysates (30 µg) were analysed with the indicated antibodies. b, As in a, with additional controls, cells treated with IFN only. Several interferon-stimulated genes were analysed. c, hTert-immortalized fibroblasts from patient P1 transduced with lentiviral particles expressing RFP and luciferase and wild-type ISG15 (LV ISG15 WT) were stimulated with IFN-β (500 pM) for 6 h. CHX was then added for the indicated times. Cell lysates (15 µg) were analysed by western blotting. d, HEK293T cells were transfected with USP18, HA–ubiquitin and Flag–ISG15 as indicated. Two days later, cells were lysed in modified RIPA buffer, USP18 was immunoprecipitated (IP) and analysed with anti-USP18 antibodies. Left panels, cell lysates (30 µg) were analysed by western blot with the indicated antibodies. Right panels, the immunoprecipitates were gel separated and transferred onto a membrane. The membrane was cut into two parts above the 50 kDa marker, both of which were blotted with anti-USP18 antibodies. The top part was exposed for 2 min, the bottom part for 20 s. Asterisk indicates IgG heavy chain. e, HEK293T cells were transfected with 1 µg of the USP18 construct alone or with 1 µg of HA– ubiquitin, in the presence or absence of Flag-tagged ISG15, either wild type or a mutant form of ISG15 that cannot be conjugated as it lacks the two carboxyterminal glycine residues (Flag–ISG15(ΔGG)). Two days later, cells were lysed in modified RIPA buffer, USP18 was immunoprecipitated and analysed with anti-HA or anti-USP18 antibodies. Asterisk indicates IgG heavy chain. f, hTert-immortalized fibroblasts from patient P3 were transfected with control siRNA or SKP2 siRNA. We added IFN-β (500 pM) 24 h later and the cells were incubated for the indicated times. Cell lysates were analysed with the indicated antibodies and USP18 levels were determined as a function of actin levels.