Fig. 1.

Identification and purification of ISG15-associated and/or -modified proteins. (A) Schematic representation of the ISG15 with two copies of FLAG at the N terminus. Mature ISG15 terminates in a conserved GG (bold), and the arrow indicates the cleavage site. ISG15 protein contains two ubiquitin-like domains and is therefore predicted to be ≈15 kDa. Indeed, ISG15 protein runs as 15 kDa in SDS/PAGE. (B–E) Validation of ISG15 conjugation of destrin, cofilin, enolase, and GAPDH. HeLa cells were transfected with plasmids encoding FLAG–ISG15 and treated with IFN-β for 48 h. Total lysates were immunoprecipitated with anti-FLAG M2-conjugated agarose and immunoblotted with antibodies against destrin, cofilin, enolase, and GAPDH.