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

Fig. 2. Two ISG15 E3 ligases HERC5 and EFP promote CHIP ISGylation.

Fig. 2

a–e All samples were transfected with plasmids encoding UBE1L and Myc-tagged UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP and/or FLAG-HERC5. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by immunoblotting with anti-Xpress or anti-HERC5 antibody. b Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Myc-EFP, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Myc or anti-Xpress antibody. c HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or its catalytically inactive mutant FLAG-HERC5-CA, alone or in combination. Cell lysates were subjected to NTA pull-down (PD: NTA) under denaturing conditions followed by western blotting with anti-FLAG or anti-Xpress antibody. d Cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, Myc-EFP-WT, or Myc-tagged catalytically inactive EFP mutant Myc-EFP-CS, alone or in combination. Cell lysates were subjected to NTA pull-down, as in c. e HEK293 cells were transfected for 48 h with nonspecific control scrambled siRNA (NC), HERC5-siRNA (H5), or plasmid encoding Xpress-His-CHIP or FLAG-ISG15, alone or in combination. Cell lysates were subjected to NTA pull-down as in b. f Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Flag-HHARI, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Flag or anti-Xpress antibody. Asterisk indicates IgG heavy chains, and closed arrowhead indicates predicted or right size band