Fig. 1.

Purification of ISG15 target proteins. (A) Establishing the conditions for optimal conjugation of plasmid-expressed tagged ISG15. HeLa cells were cotransfected with plasmids expressing His₆-HA-ISG15, E1^ISG15, and UbcH8, either without (lane 1) or with (lane 2) IFN-β treatment 24 h posttransfection. After another 24 h, cells were collected, and proteins were analyzed by immunoblotting with anti-HA antibody (Covance). (B) Scheme for purification of His₆–3xFLAG-ISG15 conjugates from 10⁸ HeLa cells. (C) Coomassie blue-stained gel of the proteins purified from IFN-β-treated cells transfected with plasmids expressing His₆–3xFLAG-ISG15, E1^ISG15, and UbcH8 (lane 2) and from untransfected, IFN-β-treated cells (lane 1).