FIG. 7.

Detection of ISG15 conjugation to specific target proteins. Plasmid constructs for ISG15 targets (pCAGGS-PLCγ1, pFlagCMV2-serpin 2A, pCEP-HA-Erk1, or pFlagCMV2-AML1B) were transfected to 293T cells in the presence or absence of a protein ISGylation system (6His-mISG15, HA-hUBE1L, and Flag-UbcM8). Cells were harvested 48 h after transfection and analyzed either by direct Western blotting (A) or by reciprocal pull-down assay followed by Western blotting (B, C, D, and E). Plasmids are identified by the ending letters and numbers of each designation. Conj., conjugate; IgG, immunoglobulin G; WB, Western blot; PD, pull-down assay; IP; immunoprecipitation.