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. 2018 Jan 24;9(2):97. doi: 10.1038/s41419-017-0138-9

Fig. 1. CHIP is a target of ISGylation in mammalian cells.

Fig. 1

a A549 cells were treated for 48 h with vehicle (−) or interferon-α (1000 U/ml). Immunoprecipitation (IP) of cell lysates was carried out with anti-CHIP antibody followed by western blotting (WB) with anti-ISG15 or anti-CHIP antibody. As a negative control, cell lysates were immunoprecipitated with preimmune IgG (IgG). The presence of ISG15 and CHIP in cell extracts was determined by western blotting with the respective antibodies. Tubulin served as a loading control. Asterisk indicates IgG heavy chains. Closed and open arrowheads indicate two specific bands of ISGylated CHIP and one non-specific background band, respectively. b HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15-WT (FLAG-ISG15-GG), or its conjugation-defective mutant (FLAG-ISG15-AA). Cell lysates were subjected to NTA pull-down (PD: NTA) under denaturing conditions, followed by western blotting with anti-FLAG or anti-Xpress antibody. Expression of Xpress-CHIP and FLAG-ISG15 was verified with anti-Xpress or anti-FLAG antibody. All samples were transfected with plasmids encoding ISG15-activating enzyme UBE1L (E1) and Myc-labeled ISG15-conjugating enzyme UbcH8 (E2). Tubulin served as a protein loading control. c HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or FLAG-UBP43, alone or in combination. Cell lysates were subjected to NTA pull-down under denaturing conditions, followed by western blotting with the indicated antibodies. All samples were transfected with plasmids encoding UBE1L (E1) and Myc-UbcH8 (E2)