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

Fig. 5. IFN-α treatment reduces c-Myc level via CHIP ISGylation.

Fig. 5

a, b All samples were transfected with plasmids encoding UBE1L and Myc-UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, V5-His-c-Myc, or FLAG-ISG15-GG, alone or in combination. Cell lysates were subjected to NTA pull-down, followed by western blotting with anti-FLAG, anti-V5, or anti-Xpress antibody. Tubulin served as a loading control. b HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, V5-His-c-Myc, FLAG-ISG15-WT, or FLAG-ISG15-AA, alone or in combination. Cell lysates were western blotted with the indicated antibodies. Values below top panel indicate the intensity of c-Myc bands relative to that of loading control α-actin. c (Upper) Schematic representation of recombinant Xpress-ISG15-CHIP fusion protein. (Lower) HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-ISG15, Xpress-His-CHIP, Xpress-His-ISG15-CHIP, or HA-c-Myc, alone or in combination. Cell lysates were western blotted with the indicated antibodies. Tubulin served as loading control. d HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP-WT, Xpress-His-CHIP-4KR, HA-c-Myc, or FLAG-ISG15, alone or in combination. All samples were transfected with plasmids encoding UBE1L and Myc-UbcH8. Cell lysates were western blotted with the indicated antibodies. Values below top panel indicate the intensity of c-Myc bands relative to that of loading control α-actin. e, f A549 cells were transfected for 24 h with nonspecific control siRNA (NC, e), CHIP-siRNA (CHIP, e), or plasmid encoding HA-CHIP-WT or HA-CHIP-4KR f, alone or in combination. Cells were treated for an additional 36 h with vehicle (−) or IFN-α (2000 U/ml). Cell lysate were then western blotted with the indicated antibodies