HyperISGylation of Old World Monkey ISG15 in Human Cells (original) (raw)
Figure 7
Promiscuity of the Activating enzyme of Ub in activating AgmISG15.
(A) In vitro assay revealing the capability of AgmISG15 to form thiolester bonds with UbE1, contrary to HuISG15. 2.5 µM UbcH8 was incubated with 200 nM of either UbE1 or UbE1L, and 7.5 µM of either mature Hu- or AgmISG15 under conditions as described in Materials and Methods. The samples were divided in two aliquots; and were subjected to SDS-PAGE under non-reducing or reducing conditions. The proteins were stained with IRDye Blue protein stain and visualized using the Odyssey infrared imaging system. The extra band representing thiolester formation between UbE1 and AgmISG15 is flanked by two asterisks. These thiolester bonds are disrupted by the addition of reducing agent (here β-ME). (B) UbE1 can efficiently activate AgmISG15, but not Hu or MoISG15. HekT cells were transfected with an empty vector, or combinations of vectors encoding HuUbE1L, HuUbE1 and UbcH8 together with HuISG15, AgmISG15 or MoISG15 as indicated. The total ISGylation patterns were evaluated by Western blot analysis using the FLAG tag of the ISG15 proteins. Equal loading was confirmed by actin staining. (C) Same as (B), but the experiment was performed in COS cells. Only co-transfections with UbcH8 are shown.